ITRM20120480A1 - MULTICOMPONENT LIPID NANOPARTICLES AND PROCEDURES FOR THEIR PREPARATION. - Google Patents
MULTICOMPONENT LIPID NANOPARTICLES AND PROCEDURES FOR THEIR PREPARATION. Download PDFInfo
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- ITRM20120480A1 ITRM20120480A1 IT000480A ITRM20120480A ITRM20120480A1 IT RM20120480 A1 ITRM20120480 A1 IT RM20120480A1 IT 000480 A IT000480 A IT 000480A IT RM20120480 A ITRM20120480 A IT RM20120480A IT RM20120480 A1 ITRM20120480 A1 IT RM20120480A1
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- Prior art keywords
- lipid
- nucleic acid
- cationic
- nanoparticles
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Classifications
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Description
"NANOPARTICELLE LIPIDICHE MULTICOMPONENTI E PROCEDIMENTI PER LA "MULTICOMPONENT LIPID NANOPARTICLES AND PROCEDURES FOR THE
LORO PREPARAZIONE" THEIR PREPARATION "
DESCRIZIONE DESCRIPTION
La presente invenzione riguarda il campo della terapia genica, in particolare riguarda nuove nano particelle lipidiche multicomponenti per il trasporto non virale di acidi nucleici e procedimenti per la loro preparazione e loro uso nella terapia genica. The present invention relates to the field of gene therapy, in particular it relates to new multicomponent lipid nano particles for the non-viral transport of nucleic acids and processes for their preparation and their use in gene therapy.
STATO DELLA TECNICA ANTERIORE STATE OF THE PRIOR ART
La terapia genica consiste nel trasferimento di materiale genetico all’interno di una cellula allo scopo di alterarne transitoriamente o permanentemente il fenotipo. Offre nuove possibilità di trattamento per numerose patologie, sia ereditarie che acquisite, che le procedure cliniche convenzionali non sono in grado di curare efficacemente. E’ ritenuta perciò la terapia del futuro e negli ultimi anni à ̈ stata oggetto d’intenso studio e sviluppo per un suo possibile impiego su larga scala in ambito clinico (“Clinical development of liposome-based drugs: formulation, characterization, and therapeutic efficacy†. Chang, H.; Yeh, M. Int. J. Nanomed. 2012, 7, 49-60). Il primo passo consiste nell’identificazione del singolo gene o dei diversi geni responsabili della malattia genetica. Ciò à ̈ possibile grazie al grande progresso delle metodiche di biologia molecolare sviluppatesi a partire dagli anni '80. Tali tecniche consentono il clonaggio e il sequenziamento di vari geni. Ciò ha comportato la precisa identificazione di molte alterazioni geniche in diverse patologie e la capacità , grazie alle tecniche del DNA ricombinante, di modificare microorganismi (come batteri o funghi) per poter far loro esprimere molecole d'interesse. Il passo successivo consiste nella valutazione della possibilità di trasfettare le cellule somatiche di un individuo avente una malattia genetica con un segmento di DNA contenente l'allele sano. Quest’approccio si à ̈ successivamente esteso anche a patologie come tumori, infezioni da HIV e altre patologie in cui non si va a sostituire un gene difettoso ma se ne aggiunge uno che possa mettere in moto un fenomeno terapeuticamente utile. Gene therapy consists in the transfer of genetic material inside a cell in order to temporarily or permanently alter its phenotype. It offers new treatment options for numerous diseases, both inherited and acquired, which conventional clinical procedures cannot effectively cure. It is therefore considered the therapy of the future and in recent years it has been the subject of intense study and development for its possible use on a large scale in the clinical setting (â € œClinical development of liposome-based drugs: formulation, characterization, and therapeutic efficacyâ €. Chang, H .; Yeh, M. Int. J. Nanomed. 2012, 7, 49-60). The first step consists in identifying the single gene or the different genes responsible for the genetic disease. This is possible thanks to the great progress of molecular biology methods developed since the 1980s. These techniques allow the cloning and sequencing of various genes. This entailed the precise identification of many gene alterations in different pathologies and the ability, thanks to recombinant DNA techniques, to modify microorganisms (such as bacteria or fungi) in order to make them express molecules of interest. The next step is to evaluate the possibility of transfecting the somatic cells of an individual with a genetic disease with a segment of DNA containing the healthy allele. This approach was subsequently extended to diseases such as tumors, HIV infections and other pathologies in which a defective gene is not replaced but one is added that can set in motion a therapeutically useful phenomenon.
Componente decisiva del trasporto genico consiste nella veicolazione del DNA all’interno delle cellule bersaglio mediante vettori che lo proteggano dalla degradazione e ne assicurino la trascrizione. I vettori utilizzati sono attualmente distinti in virali e non virali. I virus hanno un'ottima tendenza ad infettare le cellule e ad inserirvi il proprio DNA sia integrandolo sia sotto forma d'episoma. Di contro, i vettori virali presentano alcuni inconvenienti che ne limitano l’utilizzo come l'attivazione del sistema immunitario, la tossicità e la taglia ridotta del materiale genetico trasportabile (abitualmente < 40.000 coppie di basi). Quest’ultima proprietà à ̈ molto limitante considerando che molti importanti geni umani hanno una lunghezza maggiore di 40.000 coppie di basi se si considerano anche le sequenze regolatrici e gli introni non codificanti. Tra i vettori non-virali, i liposomi cationici, vescicole a doppio strato lipidico costituite di lipidi cationici e lipidi neutri, rappresentano un’attraente opportunità per l’ingegneria molecolare dal momento che si configurano come veri e propri biomateriali auto-assemblanti in scala micro/nanometrica (Elsabahy et al. “Non-Viral Nucleic Acid Delivery: Key Challenges and Future Directions†. Current Drug Delivery, 8, 2011, 235-244). In particolare, i complessi liposomi cationici-DNA (lipoplessi) rappresentano i materiali a base lipidica più diffusi. I lipoplessi presentano alcuni considerevoli vantaggi rispetto ai vettori virali quali, ad esempio, facilità di preparazione su larga scala, bassa tossicità , virtualmente nessun limite sulla taglia del DNA trasportabile. Quest’ultimo aspetto à ̈ significativo nell’ipotesi di trasferire all’interno del nucleo della cellula interi cromosomi (la dimensione media dei cromosomi umani à ̈ compresa tra 50 e 280 milioni di coppie di basi). A dispetto dei suddetti vantaggi, la limitazione principale dei lipoplessi consiste nella bassa efficienza di trasferimento del materiale genetico (efficienza di trasfezione, TE). Ad oggi, l’approccio primario per aumentare l’efficienza di trasfezione dei lipoplessi à ̈ la sintesi di nuove molecole anfipatiche o l’utilizzo di specie lipidiche non cationiche adiuvanti. Recentemente, le nanoparticelle lipidiche stanno emergendo come potenziale alternativa ai lipoplessi poiché presentano proprietà chimico-fisiche (dimensioni, carica superficiale, abilità a condensare il materiale genetico etc.) più facilmente controllabili. Tali particelle sono costituite di un nucleo di DNA (o short-interfering RNA, siRNA) condensato mediante cationi divalenti (Ca2+, Mg2+, Mn2+; Fe2+), poli-cationi (spermine, spermidine), amminoacidi cationici (arginina, lisina, istidina) e proteine cationiche (protamina, istoni) rivestito da un guscio lipidico costituito da uno ad alcuni doppi strati lipidici. La metodica di preparazione à ̈ consolidata e consiste di due passaggi successivi: (i) la formazione del nucleo di DNA/agente condensante; (ii) il successivo ricoprimento del nucleo DNA/agente condensante con un guscio lipidico. La condensazione del DNA avviene a seguito dell'interazione elettrostatica tra le cariche negative del DNA e le cariche positive del condensante cationico. Al variare del rapporto di mescolamento tra il DNA e l'agente condensante à ̈ possibile ottenere nuclei DNA/agente condensante con cariche diverse. Ciò consente di ricoprire il nucleo DNA/agente condensante con gusci lipidici che variano a seconda della carica del suddetto nucleo. A decisive component of gene transport consists in the conveyance of the DNA inside the target cells by means of vectors that protect it from degradation and ensure its transcription. The vectors used are currently divided into viral and non-viral. Viruses have an excellent tendency to infect cells and insert their DNA into them both by integrating it and in the form of an episome. On the other hand, viral vectors have some drawbacks that limit their use such as the activation of the immune system, toxicity and the reduced size of the transportable genetic material (usually <40,000 base pairs). This last property is very limiting considering that many important human genes are longer than 40,000 base pairs if we also consider the regulatory sequences and non-coding introns. Among the non-viral vectors, cationic liposomes, vesicles with a double lipid layer made up of cationic lipids and neutral lipids, represent an attractive opportunity for molecular engineering since they are configured as real self-assembling biomaterials in micro / nanoscale (Elsabahy et al. â € œNon-Viral Nucleic Acid Delivery: Key Challenges and Future Directionsâ €. Current Drug Delivery, 8, 2011, 235-244). In particular, cationic liposome-DNA complexes (lipoplexes) represent the most common lipid-based materials. Lipoplexes have some considerable advantages over viral vectors such as, for example, ease of large-scale preparation, low toxicity, virtually no limit on the size of the transportable DNA. This last aspect is significant in the hypothesis of transferring entire chromosomes into the nucleus of the cell (the average size of human chromosomes is between 50 and 280 million base pairs). In spite of the above advantages, the main limitation of lipoplexes is the low transfer efficiency of the genetic material (transfection efficiency, TE). To date, the primary approach to increase the transfection efficiency of lipoplexes is the synthesis of new amphipathic molecules or the use of adjuvant non-cationic lipid species. Recently, lipid nanoparticles are emerging as a potential alternative to lipoplexes as they have chemical-physical properties (size, surface charge, ability to condense genetic material, etc.) that are easier to control. These particles consist of a nucleus of DNA (or short-interfering RNA, siRNA) condensed by divalent cations (Ca2 +, Mg2 +, Mn2 +; Fe2 +), poly-cations (spermine, spermidine), cationic amino acids (arginine, lysine, histidine) and cationic proteins (protamine, histones) coated by a lipid shell consisting of one to a few lipid double layers. The preparation method is consolidated and consists of two successive steps: (i) the formation of the DNA nucleus / condensing agent; (ii) the subsequent coating of the DNA core / condensing agent with a lipid shell. The condensation of DNA occurs as a result of the electrostatic interaction between the negative charges of the DNA and the positive charges of the cationic condenser. By varying the mixing ratio between the DNA and the condensing agent, it is possible to obtain DNA / condensing agent nuclei with different charges. This allows to cover the DNA / condensing agent nucleus with lipid shells which vary according to the charge of the aforementioned nucleus.
La carica netta della superficie delle nanoparticelle consente una funzionalizzazione non covalente mediante interazione elettrostatica con ligandi di carica opposta. L’attività delle nanoparticelle lipidiche à ̈ strettamente connessa alle specifiche proprietà chimico-fisiche della formulazione adottata. The net charge of the surface of the nanoparticles allows a non-covalent functionalization by electrostatic interaction with ligands of opposite charge. The activity of the lipid nanoparticles is strictly connected to the specific chemical-physical properties of the formulation adopted.
Esistono numerose pubblicazioni nel campo delle nanoparticelle lipidiche, come ad esempio le pubblicazioni del gruppo del dr. L. Zhang dell’università della California a San Diego (UCSD). There are numerous publications in the field of lipid nanoparticles, such as the publications of the group of dr. L. Zhang of the University of California at San Diego (UCSD).
Nella pubblicazione “Programmed packaging of multicomponent envelope-type nanoparticle system for gene delivery†Daniela Pozzi, Carlotta Marianecci, Maria Carafa, Cristina Marchini, Maura Montani, Augusto Amici and Giulio Caracciolo. Appl. Phys. Lett.96, 183702 (2010); doi: 10.1063/1.3427354 si dimostra che una procedura di “impacchettamento programmato†consente di ottenere nanoparticelle lipidiche di dimensioni e carica controllate. I lipidi utilizzati sono lipidi anionici. In the publication â € œProgrammed packaging of multicomponent envelope-type nanoparticle system for gene deliveryâ € Daniela Pozzi, Carlotta Marianecci, Maria Carafa, Cristina Marchini, Maura Montani, Augusto Amici and Giulio Caracciolo. Appl. Phys. Lett. 96, 183702 (2010); doi: 10.1063 / 1.3427354 it is shown that a â € œprogrammed packagingâ € procedure allows to obtain lipid nanoparticles of controlled size and charge. The lipids used are anionic lipids.
Nella pubblicazione “Factors Determining the Superior Performance of Lipid/DNA/Protammine Nanoparticles over Lipoplexes†Giulio Caracciolo, Daniela Pozzi, Anna Laura Capriotti, Carlotta Marianecci, Maria Carafa, Cristina Marchini, Maura Montani, Augusto Amici, Heinz Amenitsch, Michelle A. Digman, Enrico Gratton, Susana S. Sanchez, and Aldo Laganà . J. Med. Chem. 54 (12), pp 4160–4171(2011) sono descritte nanoparticelle costituite di una singola specie lipidica cationica (DOTAP/protamina/DNA). In questa stessa pubblicazione sono discussi i fattori che determinano la maggiore efficienza delle nanoparticelle lipidiche di DOTAP rispetto ai lipoplessi DOTAP/DNA. In the publication â € œFactors Determining the Superior Performance of Lipid / DNA / Protammine Nanoparticles over Lipoplexesâ € Giulio Caracciolo, Daniela Pozzi, Anna Laura Capriotti, Carlotta Marianecci, Maria Carafa, Cristina Marchini, Maura Montani, Augusto Amici, Heinz Amenitsch, Michelle A. Digman, Enrico Gratton, Susana S. Sanchez, and Aldo LaganÃ. J. Med. Chem. 54 (12), pp 4160â € “4171 (2011) nanoparticles are described consisting of a single cationic lipid species (DOTAP / protamine / DNA). In this same publication, the factors that determine the higher efficiency of DOTAP lipid nanoparticles compared to DOTAP / DNA lipoplexes are discussed.
In questo settore in continuo sviluppo, ma passibile ancora di ampi miglioramenti, il problema principale riscontrato dai ricercatori consiste nell’individuazione di un nanovettore in grado di superare efficientemente le barriere della trasfezione cellulare. Nell’ottica della commercializzazione e di un utilizzo medico che possa essere approvato e standardizzato, un nanovettore deve presentare le seguenti caratteristiche: (i) dimensioni e carica controllate; (ii) internalizzazione cellulare efficace; (iii) rilascio citoplasmatico del carico genetico completo; (iv) efficiente ingresso nel nucleo; (v) elevata efficienza di trasfezione; (vi) elevata vitalità cellulare. In this continuously developing sector, but still subject to extensive improvements, the main problem encountered by the researchers consists in identifying a nanocarrier capable of efficiently overcoming the barriers of cell transfection. With a view to marketing and medical use that can be approved and standardized, a nano-carrier must have the following characteristics: (i) controlled size and charge; (ii) effective cell internalization; (iii) cytoplasmic release of the complete genetic load; (iv) efficient entry into the nucleus; (v) high transfection efficiency; (vi) high cell viability.
SOMMARIO DELL'INVENZIONE SUMMARY OF THE INVENTION
Al fine di risolvere il problema tecnico sopra indicato gli inventori hanno analizzato numerose formulazioni di nano particelle e hanno individuato una specifica formulazione di nanoparticelle più efficiente dei reagenti comunemente utilizzati per la trasfezione a base lipidica attualmente in commercio. E’ stata infatti realizzata una formulazione lipidica a multicomponenti che incorpori i vantaggi di ogni specie lipidica. Sono infatti oggetto della presente invenzione una nanoparticella lipidica multicomponente consistente in un nucleo costituito da una o più molecole di acido nucleico e da uno o più agenti condensanti di acidi nucleici e un rivestimento esterno costituito dai lipidi cationici 1,2-dioleoil-3-trimetilammoniopropano (DOTAP), 3beta-[N-(N’,N’-dimetilamminoetano)-carbamoil] colesterolo cloridrato (DC-Chol) e dai fosfolipidi dioleoilfosfatidiletanolammina (DOPE) e dioleilfosfocoliina (DOPC); un procedimento per la preparazione di nanoparticelle lipidiche multicomponente comprendente i seguenti passaggi: In order to solve the technical problem indicated above, the inventors have analyzed numerous formulations of nano particles and have identified a specific formulation of nanoparticles more efficient than the reagents commonly used for lipid-based transfection currently on the market. In fact, a multicomponent lipid formulation has been created that incorporates the advantages of each lipid species. The object of the present invention is in fact a multicomponent lipid nanoparticle consisting of a nucleus consisting of one or more nucleic acid molecules and one or more condensing agents of nucleic acids and an external coating consisting of cationic lipids 1,2-dioleoyl-3-trimethylammoniumopropane (DOTAP), 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and from the phospholipids dioleoylphosphatidylethanolamine (DOPE) and dioleylphosphocoliin (DOPC); a process for the preparation of multicomponent lipid nanoparticles comprising the following steps:
a. molecole di acidi nucleici ed un insieme di uno o più agenti condensanti gli acidi nucleici sono sciolti separatamente ad uguale concentrazione in H2O distillata e sono quindi miscelati tra loro ad un rapporto compreso tra 1:1 e 1:0,5 ottenendo così un complesso carico negativamente (Per lieve agitazione); to. nucleic acid molecules and a set of one or more condensing agents the nucleic acids are dissolved separately at the same concentration in distilled H2O and are then mixed together at a ratio between 1: 1 and 1: 0.5 thus obtaining a complex negatively charged (For slight agitation);
b. un volume desiderato del complesso ottenuto al punto a. Ã ̈ lasciato equilibrare per un periodo compreso tra 30 e 180 minuti a temperatura ambiente; b. a desired volume of the complex obtained in step a. It is left to equilibrate for a period between 30 and 180 minutes at room temperature;
c. si preparano liposomi cationici multicomponente sciogliendo individualmente i lipidi cationici 1,2-dioleoil-3-trimetilammoniopropano (DOTAP), 3beta-[N-(N’,N’-dimetilamminoetano)-carbamoil] colesterolo cloridrato (DC-Chol) e i fosfolipidi dioleoilfosfatidiletanolammina (DOPE) e dioleilfosfocoliina (DOPC) in un opportuno solvente organico e si miscelano quantità desiderate di ciascun lipide così sciolto fino ad ottenere una miscela omogenea; c. multicomponent cationic liposomes are prepared by individually dissolving the cationic lipids 1,2-dioleoyl-3-trimethylammoniumopropane (DOTAP), 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) and dioleylphosphocoliin (DOPC) phospholipids in a suitable organic solvent and desired quantities of each dissolved lipid are mixed until a homogeneous mixture is obtained;
d. si rimuove completamente il solvente organico dalla miscela ottenuta al punto c. ottenendo così un film lipidico; d. the organic solvent is completely removed from the mixture obtained at point c. thus obtaining a lipid film;
e. il film lipidico ottenuto al punto d. Ã ̈ idratato con un opportuno tampone a pH fisiologico cellulare fino ad ottenere una concentrazione finale desiderata; And. the lipid film obtained in point d. It is hydrated with a suitable buffer at cellular physiological pH until a desired final concentration is obtained;
f. la soluzione lipidica ottenuta al punto e. Ã ̈ sonicata fino ad ottenere una soluzione limpida (che indica la formazione di vescicole uni lamellari qui definite anche come liposomi cationici); f. the lipid solution obtained in point e. It is sonicated until a clear solution is obtained (which indicates the formation of uni-lamellar vesicles here also referred to as cationic liposomes);
g. il complesso equilibrato ottenuto al punto b. à ̈ fatto interagire con un opportuno volume della soluzione ottenuta al punto f. ad un rapporto di carica rho (Ï ) mole di lipide cationico/base di acido nucleico di compreso tra 2 e 3 estremi inclusi ed à ̈ incubato a circa 37°C fino a formazione delle nano particelle e dette nanoparticelle per uso nella terapia genica, o un metodo di terapia genica comprendente un passaggio di somministrazione di dette nanoparticelle. g. the balanced complex obtained in point b. It is made to interact with an appropriate volume of the solution obtained at point f. at a charge ratio rho (Ï) mole of cationic lipid / nucleic acid base of between 2 and 3 extremes inclusive and is incubated at about 37 ° C until the formation of nano particles and called nanoparticles for use in gene therapy, or a gene therapy method comprising a step for administering said nanoparticles.
Gli inventori hanno dimostrato (dati riportati nella sezione sperimentale) che, rispetto ai più diffusi reagenti per la trasfezione cellulare esistenti sul mercato (Lipofectamine, Lipofectamine 2000, Lipofectamine Plus) le nanoparticelle lipidiche a multicomponenti oggetto della presente invenzione evidenziano numerosi vantaggi relativamente ai problemi indicati sopra. I principali vantaggi sono i seguenti: The inventors have shown (data reported in the experimental section) that, compared to the most common reagents for cell transfection existing on the market (Lipofectamine, Lipofectamine 2000, Lipofectamine Plus), the multicomponent lipid nanoparticles object of the present invention show numerous advantages relative to the problems indicated over. The main advantages are the following:
(i) Le nanoparticelle hanno dimensioni controllate ed estremamente monodisperse; (i) The nanoparticles have controlled dimensions and extremely monodisperse;
(ii) La carica superficiale delle nanoparticelle à ̈ positiva ed estremamente riproducibile; (ii) The surface charge of the nanoparticles is positive and extremely reproducible;
(iii) L’internalizzazione cellulare à ̈ veloce ed estremamente efficace; (iii) Cell internalization is fast and extremely effective;
(iv) Il rilascio citoplasmatico del carico genetico à ̈ completo; (iv) The cytoplasmic release of the genetic load is complete;
(v) Il materiale genetico rilasciato entra efficacemente nel nucleo; (v) The released genetic material effectively enters the nucleus;
(vi) L'efficienza di trasfezione della nanoparticelle à ̈ superiore a quella dei reagenti comunemente utilizzati nella lipotrasfezione (come Lipofectamine, Lipofectamine 2000, Lipofectamine Plus) (≈ 10<10>RLU/mg proteina) in diverse linee cellulari (NIH 3T3; HeLa; CHO; A17). (vi) The transfection efficiency of the nanoparticles is higher than that of the reagents commonly used in lipotransfection (such as Lipofectamine, Lipofectamine 2000, Lipofectamine Plus) (â ‰ ˆ 10 <10> RLU / mg protein) in different cell lines (NIH 3T3; HeLa; CHO; A17).
(vii) Le nanoparticelle non sono tossiche (vitalità cellulare > 70%). (vii) The nanoparticles are not toxic (cell viability> 70%).
GLOSSARIO GLOSSARY
Per Nanoparticelle lipidiche ai fini della presente invenzione s’intendono particelle costituite da un nucleo di acidi nucleici rivestito da uno o più strati lipidici (guscio lipidico) idonee all’uso in terapia genica. By lipid nanoparticles for the purposes of the present invention we mean particles consisting of a nucleus of nucleic acids coated with one or more lipid layers (lipid shell) suitable for use in gene therapy.
Con il termine agente condensante gli acidi nucleici s’intendono molecole a carica positiva comunemente note per la loro capacità di fare condensare gli acidi nucleici, dotati di carica positiva, a questa categoria appartengono diverse classi di sostanze note in letteratura come descritto nello stato della tecnica. With the term condensing agent nucleic acids are meant positively charged molecules commonly known for their ability to condense nucleic acids, endowed with positive charge, to this category belong different classes of substances known in literature as described in the state of technique.
Per Lipidi ai fini della presente invenzione s’intendono sia lipidi come comunemente intesi nei dizionari scientifici, sia loro derivati come ad esempio derivati del colesterolo e fosfolipidi. By Lipids for the purposes of the present invention we mean both lipids as commonly understood in scientific dictionaries, and their derivatives such as cholesterol derivatives and phospholipids.
Come acidi nucleici s’intendono: dsDNA (double strand DNA), ssDNA (single strand DNA), RNA, siRNA (small interfering RNA), shRNA (short hairpin RNA), mRNA, dsRNA (double strand RNA), tRNA, miRNA (microRNA) o una loro miscela. Tali acidi nucleici potranno essere a singolo e/o doppio filamento, sotto forma di vettori integrabili nel genoma, trasponibili, minicromosomi (ovvero dotati di centromero e telomeri o più in generale, di elementi che ne consentano una replicazione autonoma), provvisti di un’origine di replicazione eccetera. As nucleic acids we mean: dsDNA (double strand DNA), ssDNA (single strand DNA), RNA, siRNA (small interfering RNA), shRNA (short hairpin RNA), mRNA, dsRNA (double strand RNA), tRNA, miRNA (microRNA) or a mixture thereof. These nucleic acids may be single and / or double-stranded, in the form of vectors that can be integrated into the genome, transposable, minichromosomes (that is, with centromere and telomeres or more generally, with elements that allow autonomous replication), provided with a ... ™ origin of replication etc.
Per trasporto genico s’intendono quelle metodologie che permettono di trasferire un acido nucleico esogeno all’interno di una cellula allo scopo di alterarne transitoriamente o permanentemente il fenotipo come ad esempio tecniche di trasfezione che permettono di esprimere o silenziare specifici geni nelle cellule trasformate. By gene transport we mean those methodologies that allow to transfer an exogenous nucleic acid inside a cell in order to temporarily or permanently alter its phenotype such as transfection techniques that allow to express or silence specific genes in transformed cells .
Il termine silenziamento genico à ̈ utilizzato nella presente descrizione in accordo con lo stato della tecnica. The term gene silencing is used in the present description in accordance with the state of the art.
Per terapia genica s’intende l'inserzione di materiale genetico come sopra definito (acidi nucleici) all'interno di cellule al fine di poter curare delle patologie. Questa procedura d’inserzione, nota come trasfezione, permette di trasferire uno o più geni sani in una cellula malata, al fine di curare una patologia causata dall'assenza o dal difetto di uno o più geni (mutati) o di uno o più acidi nucleici in grado di silenziare geni che sono sovra espressi o espressi nella cellula malata. Gene therapy means the insertion of genetic material as defined above (nucleic acids) inside cells in order to be able to cure pathologies. This insertion procedure, known as transfection, allows the transfer of one or more healthy genes into a diseased cell, in order to treat a disease caused by the absence or defect of one or more (mutated) genes or of one or more nucleic acids capable of silencing genes that are over-expressed or expressed in the diseased cell.
L’acronimo DOTAP indica il lipide cationico 1,2-dioleoil-3-trimetilammoniopropano. L’acronimo DC-Chol indica il lipide cationico derivato del colesterolo 3beta-[N-(N’,N’-dimetilamminoetano)-carbamoil] colesterolo cloridrato. The acronym DOTAP indicates the cationic lipid 1,2-dioleoyl-3-trimethylammoniumopropane. The acronym DC-Chol indicates the cationic lipid derived from cholesterol 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride.
L’acronimo DOPE indica il fosfolipide dioleoilfosfatidiletanolammina. The acronym DOPE indicates the phospholipid dioleoylphosphatidylethanolamine.
L’acronimo DOPC indica il fosfolipide dioleilfosfocoliina. The acronym DOPC indicates the phospholipid dioleylphosphocoliin.
DESCRIZIONE DETTAGLIATA DELLE FIGURE DETAILED DESCRIPTION OF THE FIGURES
La figura 1 mostra in 1(A) l’efficienza di trasfezione nelle cellule ovariche CHO di diverse nanoparticelle secondo l’invenzione (sono indicati i rapporti relativi tra i lipidi cationici 1,2-dioleoil-3-trimetilammoniopropano (DOTAP), 3beta-[N-(N’,N’-dimetilamminoetano)-carbamoil] colesterolo cloridrato (DC-Chol) e i fosfolipidi dioleoilfosfatidiletanolammina (DOPE) e dioleilfosfocoliina (DOPC) sotto ad ogni istogramma) e della Lipofectamine che à ̈ uno dei reagenti più comuni per la trasfezione cellulare. Gli istogrammi in grigio si riferiscono ad esperimenti effettuati utilizzando 5 µg di campione (per campione s’intende la soluzione di liposomi cationici descritti al punto f. del metodo sopra) per pozzetto, quelli in nero utilizzando 10 µg di campione per pozzetto. In 1 (B) la figura mostra l’efficienza di trasfezione nei fibroblasti NIH 3T3 di diverse nanoparticelle secondo l’invenzione (sono indicati i rapporti relativi tra i lipidi cationici 1,2-dioleoil-3-trimetilammoniopropano (DOTAP), 3beta-[N-(N’,N’-dimetilamminoetano)-carbamoil] colesterolo cloridrato (DC-Chol) e i fosfolipidi dioleoilfosfatidiletanolammina (DOPE) e dioleilfosfocoliina (DOPC) sotto ad ogni istogramma) e della Lipofectamine. Gli istogrammi in grigio si riferiscono ad esperimenti effettuati utilizzando 5 µg di campione per pozzetto, quelli in nero utilizzando 10 µg di campione per pozzetto. Figure 1 shows in 1 (A) the transfection efficiency in CHO ovarian cells of different nanoparticles according to the invention (the relative ratios between the cationic lipids 1,2-dioleoyl-3-trimethylammoniumopropane (DOTAP) are shown, 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and the phospholipids dioleoylphosphatidylethanolamine (DOPE) and dioleylphosphocoliin (DOPC) under each histogram most common reagents for cell transfection. The gray histograms refer to experiments carried out using 5 µg of sample (for sample we mean the solution of cationic liposomes described in point f. Of the above method) per well, those in black using 10 µg of sample per well. In 1 (B) the figure shows the transfection efficiency in NIH 3T3 fibroblasts of different nanoparticles according to the invention (the relative ratios between the cationic lipids 1,2-dioleoyl-3-trimethylammoniopropane (DOTAP), 3beta are shown - [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and the phospholipids dioleoylphosphatidylethanolamine (DOPE) and dioleylphosphocoliin (DOPC) under each histogram) and of Lipofectamine. The gray histograms refer to experiments carried out using 5 µg of sample per well, those in black using 10 µg of sample per well.
La figura 2 mostra i risultati di un saggio di vitalità cellulare condotto sulle cellule ovariche CHO come descritto nella sezione sperimentale (sono indicati i rapporti relativi tra i lipidi cationici 1,2-dioleoil-3-trimetilammoniopropano (DOTAP), 3beta-[N-(N’,N’-dimetilamminoetano)-carbamoil] colesterolo cloridrato (DC-Chol) e i fosfolipidi dioleoilfosfatidiletanolammina (DOPE) e dioleilfosfocoliina (DOPC) sotto ad ogni istogramma). Figure 2 shows the results of a cell viability assay conducted on CHO ovarian cells as described in the experimental section (the relative ratios between the cationic lipids 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and the phospholipids dioleoylphosphatidylethanolamine (DOPE) and dioleylphosphocoliin (DOPC) under each histogram).
La figura 3 mostra sovrapposizioni rappresentative di immagini di fluorescenza e immagini Nomarski nelle cellule CHO. Il segnale bianco à ̈ dovuto alla presenza del DNA fluorescente. Figure 3 shows representative overlays of fluorescence images and Nomarski images in CHO cells. The white signal is due to the presence of the fluorescent DNA.
Per ogni immagine à ̈ stato effettuato uno scan lungo l’asse z. Come si vede nelle due immagini rappresentative, il DNA à ̈ uniformemente diffuso nel citoplasma e nel nucleo della cellula. Rispetto a quanto che si osserva con i più diffusi reagenti per la trasfezione a base lipidica, non sono visibili aggregati perinucleari con DNA confinato. For each image a scan was performed along the z axis. As seen in the two representative images, the DNA is uniformly diffused in the cytoplasm and nucleus of the cell. Compared to what is observed with the most common lipid-based transfection reagents, no perinuclear aggregates with confined DNA are visible.
DESCRIZIONE DETTAGLIATA DELL'INVENZIONE DETAILED DESCRIPTION OF THE INVENTION
La presente invenzione riguarda dunque nano particelle lipidiche multicomponente consistente in un nucleo costituito da una o più molecole di acido nucleico e da uno o più agenti condensanti di acidi nucleici e un rivestimento esterno costituito dai lipidi cationici 1,2-dioleoil-3-trimetilammoniopropano (DOTAP), 3beta-[N-(N’,N’-dimetilamminoetano)-carbamoil] colesterolo cloridrato (DC-Chol) e dai fosfolipidi dioleoilfosfatidiletanolammina (DOPE) e dioleilfosfocoliina (DOPC). The present invention therefore relates to multicomponent lipid nano particles consisting of a nucleus consisting of one or more nucleic acid molecules and one or more condensing agents of nucleic acids and an external coating consisting of cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane ( DOTAP), 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and by the phospholipids dioleoylphosphatidylethanolamine (DOPE) and dioleylphosphocoliin (DOPC).
Secondo la presente invenzione, poiché la nanoparticella deve essere idonea all’uso in terapia genica, le molecole di acido nucleico possono essere scelte tra dsDNA (double strand DNA), ssDNA (single strand DNA) ovvero DNA a doppio o singolo filamento, RNA, siRNA (small interfering RNA), shRNA (short hairpin RNA), mRNA, dsRNA (double strand RNA), tRNA, miRNA (microRNA) o una loro miscela. According to the present invention, since the nanoparticle must be suitable for use in gene therapy, the nucleic acid molecules can be chosen from dsDNA (double strand DNA), ssDNA (single strand DNA) or double or single stranded DNA, RNA, siRNA (small interfering RNA), shRNA (short hairpin RNA), mRNA, dsRNA (double strand RNA), tRNA, miRNA (microRNA) or a mixture thereof.
Le molecole di acido nucleico potranno essere inserite in opportuni vettori che ne permettano l’incorporazione nel genoma ospite (ad esempio vettori trasponibili) o che ne permettano la replicazione e l’espressione o la trascrizione nella cellula ospite (cellula eucariote), come ad esempio opportuni vettori di clonaggio, vettori di espressione, minicromosomi eccetera. Gli acidi nucleici (anche definiti qui come “materiale genetico†in generale) potranno essere geni per terapia genica sostitutiva (cioà ̈ per terapia genica in cui la malattia à ̈ causata da una mancata espressione di un determinato gene o in cui à ̈ espresso un gene non funzionale) o potranno essere sequenze volte ad interferire con l’espressione di uno o più geni espressi o sovra espressi nelle cellule malate, come ad esempio nelle cellule tumorali, e normalmente non espressi nelle cellule sane. Il materiale genetico inserito potrà anche consistere di tRNA laddove la disfunzione sia legata all’assenza o al mal funzionamento di un particolare tRNA. The nucleic acid molecules can be inserted into suitable vectors that allow their incorporation into the host genome (for example transposable vectors) or that allow their replication and expression or transcription in the host cell (eukaryotic cell), such as for example suitable cloning vectors, expression vectors, minichromosomes, etc. Nucleic acids (also defined here as `` genetic material '' in general) may be genes for gene replacement therapy (i.e. for gene therapy in which the disease is caused by a lack of expression of a certain gene or in which it is expressed a non-functional gene) or they may be sequences aimed at interfering with the expression of one or more genes expressed or over-expressed in diseased cells, such as in cancer cells, and not normally expressed in healthy cells. The inserted genetic material may also consist of tRNA where the dysfunction is related to the absence or malfunction of a particular tRNA.
Tali acidi nucleici sono definiti anche nella presente descrizione come “sequenze di interesse†poiché nella realizzazione dell’invenzione come qui insegnata non à ̈ rilevante, ai fini dell’ottenimento di una particella avente i vantaggi tecnici sopra elencati, quali sequenze siano inserite nel nucleo acidi nucleici/agente condensante di acidi nucleici, qualsiasi sequenza potrà essere inserita senza che cambino le caratteristiche chimico-fisiche delle nano particelle. These nucleic acids are also defined in the present description as â € œsequences of interestâ € since in the realization of the invention as taught here it is not relevant, for the purposes of obtaining a particle having the technical advantages listed above, such as sequences nucleic acids / nucleic acid condensing agent are inserted in the nucleus, any sequence can be inserted without changing the chemical-physical characteristics of the nano particles.
È evidente che nella forma di realizzazione preferita tali sequenze avranno una funzione riconosciuta nella terapia genica. It is evident that in the preferred embodiment such sequences will have a recognized function in gene therapy.
Il tecnico del settore saprà “caricare†le nanoparticelle dell’invenzione con gli acidi nucleici idonei all’obiettivo medico da perseguire senza bisogno di ulteriori insegnamenti nella presente descrizione che fornisce efficaci “mezzi di trasporto†per tali acidi nucleici e procedimenti dettagliati per realizzarli. The person skilled in the art will be able to â € œfillâ € the nanoparticles of the invention with the nucleic acids suitable for the medical objective to be pursued without the need for further teaching in the present description which provides effective â € œmeans of transportâ € for such nucleic acids and procedures. detailed to make them.
Il rapporto tra il volume di dette una o più molecole di acido nucleico e di detti agenti condensanti di acidi nucleici nelle nano particelle secondo l’invenzione potrà essere compreso tra circa 1:1 e circa 1:0,5, in una forma di realizzazione tale rapporto sarà di circa 1:0,75. The ratio between the volume of said one or more nucleic acid molecules and of said nucleic acid condensing agents in the nanoparticles according to the invention may be comprised between about 1: 1 and about 1: 0.5, in a form of realization this ratio will be approximately 1: 0.75.
Per la realizzazione della presente invenzione potranno essere utilizzati uno o più condensanti di acidi nucleici comunemente noti nel settore. Ad esempio, tali agenti potranno essere scelti tra poliammine (ad esempio spermina o spermidina) , peptidi cationici (esempio?), lipidi cationici (esempio?), tensioattivi cationici (esempio?), amminoacidi a carica positiva (ad esempio arginina e lisina), proteine a carica positiva, cationi metallici multivalenti (ad esempio Ca<2+>, Mg<2+>, Mn<2+>; Fe<2+>,Fe<3+>). One or more nucleic acid condensers commonly known in the art can be used to carry out the present invention. For example, such agents can be selected from polyamines (for example spermine or spermidine), cationic peptides (for example?), Cationic lipids (for example?), Cationic surfactants (for example?), Positively charged amino acids (for example arginine and lysine) , positively charged proteins, multivalent metal cations (e.g. Ca <2+>, Mg <2+>, Mn <2+>; Fe <2 +>, Fe <3+>).
Gli esempi sopra indicati non sono limitativi della realizzazione dell’invenzione ma hanno lo scopo di fornire al lettore della presente invenzione esempi concreti di molecole idonee tra quelle note al tecnico del settore. The examples indicated above are not limitative of the realization of the invention but have the purpose of providing the reader of the present invention with concrete examples of suitable molecules among those known to those skilled in the art.
In una particolare forma di realizzazione il nucleo della nanoparticella sarà realizzato utilizzando come condensante la protammina. In a particular embodiment, the nucleus of the nanoparticle will be made using protamine as the condenser.
In questa forma di realizzazione il nucleo avrà un diametro idrodinamico, D = 230 ± 18 nm; e una carica superficiale = - 19.5 ± 2.5 mV. In this embodiment the core will have a hydrodynamic diameter, D = 230 ± 18 nm; and a surface charge = - 19.5 ± 2.5 mV.
Per quanto riguarda il guscio esterno della nanoparticella, i lipidi cationici e i fosfolipidi potranno essere in rapporti reciproci variabili, secondo alcune forme di realizzazione detti lipidi cationici 1,2-dioleoil-3-trimetilammoniopropano (DOTAP), 3beta-[N-(N’,N’-dimetilamminoetano)-carbamoil] colesterolo cloridrato (DC-Chol) e detti fosfolipidi dioleoilfosfatidiletanolammina (DOPE) e dioleilfosfocoliina (DOPC) saranno presenti in un rapporto reciproco scelto tra 1,5:0,5:1,5:0,5 o 1:1:1:1 o 0,5:1,5:0,5:1,5. As far as the outer shell of the nanoparticle is concerned, the cationic lipids and the phospholipids may be in variable reciprocal ratios, according to some embodiments called cationic lipids 1,2-dioleoyl-3-trimethylammonopropane (DOTAP), 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and said phospholipids dioleoylphosphatidylethanolamine (DOPE) and dioleylphosphocoliin (DOPC) will be present in a reciprocal ratio chosen between 1.5: 0.5: 1.5: 0.5 or 1: 1: 1: 1 or 0.5: 1.5: 0.5: 1.5.
In una particolare forma di realizzazione il rapporto reciproco sopra menzionato sarà 0,5:1,5:0,5:1,5. In a particular embodiment the above mentioned reciprocal ratio will be 0.5: 1.5: 0.5: 1.5.
Nella realizzazione della presente invenzione, data la scelta dei lipidi di rivestimento, la carica superficiale della nanoparticella à ̈ positiva e, nella forma di realizzazione in cui il rapporto reciproco tra detti lipidi cationici 1,2-dioleoil-3-trimetilammoniopropano (DOTAP), 3beta-[N-(N’,N’-dimetilamminoetano)-carbamoil] colesterolo cloridrato (DC-Chol) e detti fosfolipidi dioleoilfosfatidiletanolammina (DOPE) e dioleilfosfocoliina (DOPC) à ̈ 0,5:1,5:0,5:1,5.la carica superficiale della nanoparticella avrà un potenziale z compreso tra circa 40 e 45 mV, un esempio non limitativo à ̈ rappresentato da un potenziale z di circa 42mV. In the realization of the present invention, given the choice of the coating lipids, the surface charge of the nanoparticle is positive and, in the embodiment in which the reciprocal ratio between said cationic lipids 1,2-dioleoyl-3-trimethylammoniumopropane (DOTAP), 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and said phospholipids dioleoylphosphatidylethanolamine (DOPE) and dioleylphosphocoliin (DOPC) à ̈ 0.5: 1.5: 0, 5: 1.5 the surface charge of the nanoparticle will have a z potential between about 40 and 45 mV, a non-limiting example is represented by a z potential of about 42mV.
La nanoparticella lipidica realizzata come qui descritto, avrà diametro compreso tra 200 e 250 nm e potrà avere un indice di polidispersità <0,2. The lipid nanoparticle made as described here will have a diameter between 200 and 250 nm and may have a polydispersity index <0.2.
Nella forma di realizzazione in cui il rapporto reciproco tra detti lipidi cationici 1,2-dioleoil-3-trimetilammoniopropano (DOTAP), 3beta-[N-(N’,N’-dimetilamminoetano)-carbamoil] colesterolo cloridrato (DC-Chol) e detti fosfolipidi dioleoilfosfatidiletanolammina (DOPE) e dioleilfosfocoliina (DOPC) à ̈ 0,5:1,5:0,5:1,5 il diametro D della particella sarà di circa 210 ± 12 nm e l’indice di polidispersità sarà <0,2. In the embodiment in which the reciprocal ratio of said cationic lipids 1,2-dioleoyl-3-trimethylammoniumopropane (DOTAP), 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC- Chol) and said phospholipids dioleoylphosphatidylethanolamine (DOPE) and dioleylphosphocoliin (DOPC) is 0.5: 1.5: 0.5: 1.5 the diameter D of the particle will be about 210 ± 12 nm and the polydispersity index it will be <0.2.
Le suddette nanoparticelle presentano le seguenti caratteristiche The aforementioned nanoparticles exhibit the following characteristics
(i) Le nanoparticelle hanno dimensioni controllate ed estremamente monodisperse (indice di polidispersità < 0.2) (i) The nanoparticles have controlled dimensions and extremely monodisperse (polydispersity index <0.2)
(ii) Hanno una carica superficiale positiva e estremamente riproducibile; (ii) They have a positive and extremely reproducible surface charge;
(iii) L’internalizzazione cellulare à ̈ veloce ed estremamente efficace; (iii) Cell internalization is fast and extremely effective;
(iv) Il rilascio citoplasmatico del carico genetico à ̈ completo; (iv) The cytoplasmic release of the genetic load is complete;
(v) Il materiale genetico rilasciato entra efficacemente nel nucleo; (v) The released genetic material effectively enters the nucleus;
(vi) L'efficienza di trasfezione della nanoparticelle à ̈ superiore a quella dei reagenti comunemente utilizzati per la lipotrasfezione, come le lipofectamine (≈ 10<10>RLU/mg proteina) in diverse linee cellulari (NIH 3T3; HeLa; CHO; A17). (vi) The transfection efficiency of nanoparticles is higher than that of commonly used lipotransfection reagents, such as lipofectamines (â ‰ ˆ 10 <10> RLU / mg protein) in different cell lines (NIH 3T3; HeLa; CHO ; A17).
(vii) Le nanoparticelle non sono tossiche (vitalità cellulare > 70%). (vii) The nanoparticles are not toxic (cell viability> 70%).
La presente invenzione riguarda anche un procedimento per la preparazione di nanoparticelle lipidiche multicomponente comprendente i seguenti passaggi: The present invention also relates to a process for the preparation of multicomponent lipid nanoparticles comprising the following steps:
a. molecole di acidi nucleici ed un insieme di uno o più agenti condensanti gli acidi nucleici sono sciolti separatamente ad uguale concentrazione in H2O distillata e sono quindi miscelati tra loro ad un rapporto compreso tra 1:1 e 1:0,5 ottenendo così un complesso carico negativamente (Per lieve agitazione); b. un volume desiderato del complesso ottenuto al punto a. à ̈ lasciato equilibrare per un periodo compreso tra 30 e 180 minuti a temperatura ambiente; to. nucleic acid molecules and a set of one or more condensing agents the nucleic acids are dissolved separately at the same concentration in distilled H2O and are then mixed together at a ratio between 1: 1 and 1: 0.5 thus obtaining a complex negatively charged (For slight agitation); b. a desired volume of the complex obtained in step a. It is left to equilibrate for a period between 30 and 180 minutes at room temperature;
c. si preparano liposomi cationici multicomponente sciogliendo individualmente i lipidi cationici 1,2-dioleoil-3-trimetilammoniopropano (DOTAP), 3beta-[N-(N’,N’-dimetilamminoetano)-carbamoil] colesterolo cloridrato (DC-Chol) e i fosfolipidi dioleoilfosfatidiletanolammina (DOPE) e dioleilfosfocoliina (DOPC) in un opportuno solvente organico e si miscelano quantità desiderate di ciascun lipide così sciolto fino ad ottenere una miscela omogenea; c. multicomponent cationic liposomes are prepared by individually dissolving the cationic lipids 1,2-dioleoyl-3-trimethylammoniumopropane (DOTAP), 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) and dioleylphosphocoliin (DOPC) phospholipids in a suitable organic solvent and desired quantities of each dissolved lipid are mixed until a homogeneous mixture is obtained;
d. si rimuove completamente il solvente organico dalla miscela ottenuta al punto c. ottenendo così un film lipidico; d. the organic solvent is completely removed from the mixture obtained at point c. thus obtaining a lipid film;
e. il film lipidico ottenuto al punto d. à ̈ idratato con un opportuno tampone a pH fisiologico cellulare fino ad ottenere una concentrazione finale desiderata; f. la soluzione lipidica ottenuta al punto e. à ̈ sonicata fino ad ottenere una soluzione limpida (che indica la formazione delle vescicole uni lamellari); g. il complesso equilibrato ottenuto al punto b. à ̈ fatto interagire con un opportuno volume della soluzione ottenuta al punto f. ad un rapporto di carica rho mole di lipide cationico/base di acido nucleico compreso tra circa 2 e 3, estremi inclusi, ed à ̈ incubato a circa 37°C fino a formazione delle nanoparticelle. And. the lipid film obtained in point d. It is hydrated with a suitable buffer at cellular physiological pH until a desired final concentration is obtained; f. the lipid solution obtained in point e. It is sonicated until a clear solution is obtained (which indicates the formation of uni lamellar vesicles); g. the balanced complex obtained in point b. It is made to interact with an appropriate volume of the solution obtained at point f. at a charge ratio rho mole of cationic lipid / base of nucleic acid between about 2 and 3, extremes included, and it is incubated at about 37 ° C until nanoparticles are formed.
Nel procedimento qui descritto, la concentrazione della soluzione di acidi nucleici e della soluzione contenente l’agente o gli agenti condensanti gli acidi nucleici potranno avere una concentrazione scelta secondo le necessità dell’operatore, tale concentrazione, che sarà uguale per le due diverse soluzioni, potrà essere tra 0,5 e 1,5 mg/ml ad esempio, e cioà ̈ circa 0,5; circa 0,6; circa 0,7; circa 0,8; circa 0,9; circa 1; circa 1,1; circa 1,2; circa 1,3; circa 1,4; circa 1,5 mg/ml a titolo esemplificativo anche se la presente invenzione non esclude che il tecnico del settore possa selezionare concentrazioni diverse. In the procedure described here, the concentration of the solution of nucleic acids and of the solution containing the agent or agents condensing the nucleic acids may have a concentration chosen according to the needs of the operator, this concentration, which will be the same for the two different solutions, may be between 0.5 and 1.5 mg / ml for example, ie about 0.5; about 0.6; about 0.7; about 0.8; about 0.9; about 1; about 1.1; about 1.2; about 1.3; about 1.4; about 1.5 mg / ml by way of example, even if the present invention does not exclude that the skilled in the art can select different concentrations.
Il rapporto di volume tra la soluzione di acidi nucleici e la soluzione di agente condensante gli acidi nucleici al punto a. sarà , come descritto, in un intervallo tra 1:1 e 0,5:1. A titolo esemplificativo e non limitativo, tale rapporto potrà essere, nell’ordine sopra indicato (volume della soluzione di acidi nucleici: volume della soluzione di agenti condensanti gli acidi nucleici) circa 0,5:1: 0.55:1; 0.6:1; 065:1; 0,7:1; 0,75:1; 0,8:1; 0,85:1; 0,9:1; 0.95:1; 1:1. The volume ratio between the nucleic acid solution and the nucleic acid condensing agent solution in step a. it will be, as described, in a range between 1: 1 and 0.5: 1. By way of non-limiting example, this ratio may be, in the order indicated above (volume of the nucleic acid solution: volume of the solution of nucleic acid condensing agents) about 0.5: 1: 0.55: 1; 0.6: 1; 065: 1; 0.7: 1; 0.75: 1; 0.8: 1; 0.85: 1; 0.9: 1; 0.95: 1; 1: 1.
Le molecole di acidi nucleici che possono essere utilizzate per la preparazione del nucleo delle nanoparticelle dell’invenzione sono quelle indicate precedentemente nella parte relativa alla descrizione delle nanoparticelle stesse e potranno quindi essere scelte tra DNA, RNA, siRNA, shRNA, mRNA; miRNA; dsRNA. The nucleic acid molecules that can be used for the preparation of the nucleus of the nanoparticles of the invention are those indicated previously in the part relating to the description of the nanoparticles themselves and can therefore be chosen from DNA, RNA, siRNA, shRNA, mRNA; miRNA; dsRNA.
Nel procedimento sopra descritto, gli agenti condensanti di acidi nucleici sono scelti tra poliammine, peptidi cationici, lipidi cationici, tensioattivi cationici, amminoacidi a carica positiva, proteine a carica positiva, cationi metallici multivalenti. Esempi non limitativi di molecole idonee sono qui forniti nella parte relativa alla descrizione delle nanoparticelle stesse e potranno quindi essere: per le poliammine scelte tra spermina e spermidina, per gli amminoacidi a carica positiva scelti tra arginina e lisina, per le proteine a carica positiva scelte tra protammina e istoni, per i cationi metallici multivalenti scelti tra Ca<2+>, Mg<2+>, Mn<2+>; Fe<2+>; Fe<3+>. In the process described above, the condensing agents of nucleic acids are selected from polyamines, cationic peptides, cationic lipids, cationic surfactants, positively charged amino acids, positively charged proteins, multivalent metal cations. Non-limiting examples of suitable molecules are provided here in the part relating to the description of the nanoparticles themselves and may therefore be: for the polyamines selected from spermine and spermidine, for the positively charged amino acids selected from arginine and lysine, for the positively charged proteins selected between protamine and histones, for the multivalent metal cations selected from Ca <2+>, Mg <2+>, Mn <2+>; Fe <2+>; Fe <3+>.
In una particolare forma di realizzazione à ̈ scelta, come agente condensante di acidi nucleici, la protammina. In a particular embodiment, protamine is selected as the condensing agent of nucleic acids.
Nel procedimento sopra descritto, si lascia equilibrare un volume desiderato del complesso ottenuto in a. (che corrisponderà al “nucleo†delle nanoparticelle), per un periodo compreso tra 30 e 180 minuti circa, questo periodo potrà essere, ad esempio di 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 minuti circa. In generale à ̈ sufficiente anche un periodo compreso tra circa 30 e 60 minuti circa, estremi inclusi. In the process described above, a desired volume of the complex obtained in a is allowed to equilibrate. (which will correspond to the â € œnucleusâ € of the nanoparticles), for a period between 30 and 180 minutes approximately, this period could be, for example, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 , 130, 140, 150, 160, 170, 180 minutes approximately. In general, a period of about 30 to 60 minutes, including extremes, is also sufficient.
I nuclei così ottenuti possono essere conservati per un periodo da qualche ora a circa 24 ore a circa 4°C. The nuclei thus obtained can be stored for a period from a few hours to about 24 hours at about 4 ° C.
Poiché il nucleo ottenuto in b. contenendo acidi nucleici, à ̈ più delicato rispetto alle vescicole unilamellari ottenute al punto f., che possono essere conservate in frigorifero (a circa 4°C) per circa una settimana o due, à ̈ consigliabile preparare prima le vescicole o comunque preparare il nucleo e le vescicole contemporaneamente in modo da utilizzare il nucleo ottenuto in b. il prima possibile ed evitare possibili degradazioni dello stesso al fine di ottenere nanoparticelle multicomponente con la maggiore efficacia. Since the nucleus obtained in b. containing nucleic acids, it is more delicate than the unilamellar vesicles obtained in step f., which can be stored in the refrigerator (at about 4 ° C) for about a week or two, it is advisable to prepare the vesicles first or otherwise prepare the nucleus and the vesicles simultaneously in order to use the nucleus obtained in b. as soon as possible and avoid possible degradation of the same in order to obtain multicomponent nanoparticles with the greatest effectiveness.
Nel procedimento descritto, per la preparazione delle vescicole da utilizzare per il realizzare il rivestimento del nucleo, si utilizzeranno i lipidi (cationici e fosfolipidi) sopra indicati, nei rapporti reciproci desiderati. Si à ̈ visto che i vari rapporti saggiati sono tutti efficaci per realizzare vescicole con buone proprietà di trasfezione e bassa tossicità . In particolare, al punto c. potranno essere miscelati i lipidi cationici 1,2-dioleoil-3-trimetilammoniopropano (DOTAP), 3beta-[N-(N’,N’-dimetilamminoetano)-carbamoil] colesterolo cloridrato (DC-Chol) e i fosfolipidi dioleoilfosfatidiletanolammina (DOPE) e dioleilfosfocoliina (DOPC) in un rapporto reciproco scelto tra 1,5:0,5:1,5:0,5 o 1:1:1:1 o 0,5:1,5:0,5:1,5. In the described process, for the preparation of the vesicles to be used for the coating of the core, the above mentioned lipids (cationic and phospholipids) will be used, in the desired reciprocal ratios. It has been found that the various ratios tested are all effective in producing vesicles with good transfection properties and low toxicity. In particular, in point c. cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and the phospholipids dioleoylphosphatidylethanol ) and dioleylphosphocoliin (DOPC) in a reciprocal ratio chosen between 1.5: 0.5: 1.5: 0.5 or 1: 1: 1: 1 or 0.5: 1.5: 0.5: 1, 5.
In una forma di realizzazione particolarmente interessante, i suddetti lipidi potranno essere miscelati, e quindi presenti nel rivestimento del nucleo acido nucleico/condensante delle nanoparticelle qui descritte, nel rapporto reciproco (in cui l’ordine dei lipidi à ̈ quello indicato nel paragrafo precedente) 0,5:1,5:0,5:1,5. In a particularly interesting embodiment, the aforementioned lipids can be mixed, and therefore present in the coating of the nucleic acid / condensing core of the nanoparticles described here, in the reciprocal ratio (in which the order of the lipids is that indicated in the previous paragraph ) 0.5: 1.5: 0.5: 1.5.
Ciascun lipide sarà sciolto in un solvente organico opportuno e quindi mescolato nei rapporti desiderati o indicati sopra. Each lipid will be dissolved in a suitable organic solvent and then mixed in the desired ratios or indicated above.
Qualsiasi solvente organico idoneo potrà essere utilizzato, ad esempio, solventi organici idonei, comunemente utilizzati dal tecnico del settore, potranno essere cloroformio o etanolo. Any suitable organic solvent can be used, for example, suitable organic solvents, commonly used by the skilled in the art, can be chloroform or ethanol.
I reagenti saranno miscelati fino ad ottenere una soluzione omogenea, a temperatura ambiente per un periodo compreso tra 1 e 5 minuti, come ad esempio circa 1-2 minuti. In una forma di realizzazione dell’invenzione, I liposomi cationici a multicomponente costituiti dalle quattro specie lipidiche che si ottengono al punto f. si possono preparare ad una frazione molare di lipide neutro nel doppio strato Φ = (lipide neutro /lipide totale) (mol/mol) = tra 0.4 e 0.6, ad esempio 0.5. I lipidi si sciolgono nell’opportuno solvente, ad esempio cloroformio, e si mescolano fino ad ottenere un miscelamento omogeneo delle quattro specie lipidiche (in solvente organico il miscelamento di molecole lipidiche à ̈ un miscelamento omogeneo). La quantità di solvente da utilizzare dipende dalla solubilità delle specie lipidiche utilizzate, ad esempio, le soluzioni si possono preparare a circa 10/20 mg lipide/ml cloroformio. The reagents will be mixed until a homogeneous solution is obtained, at room temperature for a period of between 1 and 5 minutes, such as approximately 1-2 minutes. In an embodiment of the invention, the multicomponent cationic liposomes consist of the four lipid species which are obtained at point f. they can be prepared with a molar fraction of neutral lipid in the bilayer Φ = (neutral lipid / total lipid) (mol / mol) = between 0.4 and 0.6, for example 0.5. The lipids dissolve in the appropriate solvent, for example chloroform, and mix until obtaining a homogeneous mixing of the four lipid species (in organic solvent the mixing of lipid molecules is a homogeneous mixing). The amount of solvent to be used depends on the solubility of the lipid species used, for example, the solutions can be prepared at approximately 10/20 mg lipid / ml chloroform.
Il solvente organico à ̈ quindi rimosso al punto d., ciò può essere realizzato con qualsiasi opportuno mezzo noto al tecnico del settore, come ad esempio utilizzando un evaporatore rotante convenzionale. Tale procedura porta alla formazione di un sottile film lipidico sul fondo del recipiente utilizzato. La rimozione del solvente organico deve essere completa, per assicurare la completa rimozione del solvente organico, il film lipidico può essere ad esempio tenuto per 24 ore sotto vuoto. The organic solvent is then removed at point d., This can be achieved with any suitable means known to the person skilled in the art, such as using a conventional rotary evaporator. This procedure leads to the formation of a thin lipid film on the bottom of the container used. The removal of the organic solvent must be complete, to ensure the complete removal of the organic solvent, the lipid film can for example be kept for 24 hours under vacuum.
Successivamente, il film lipidico al punto d. Ã ̈ idratato con un opportuno tampone a pH fisiologico cellulare fino ad ottenere una concentrazione finale desiderata. Subsequently, the lipid film in point d. It is hydrated with a suitable buffer at cellular physiological pH until a desired final concentration is obtained.
Qualsiasi tampone comunemente idoneo all’uso con cellule vitali, che stabilizzi il pH a livelli fisiologici, come ad esempio il tampone Tris-HCl a pH fisiologico potrà esser utilizzato. Any buffer commonly suitable for use with viable cells, which stabilizes the pH at physiological levels, such as the physiological pH Tris-HCl buffer can be used.
Ad esempio potrà essere utilizzato Tris-HCl (10 mM, pH 7.4) necessario ad ottenere una concentrazione finale di 1 mg/ml. For example, Tris-HCl (10 mM, pH 7.4) can be used, necessary to obtain a final concentration of 1 mg / ml.
A questo punto, la soluzione lipidica ottenuta al punto e. sarà sonicata fino ad ottenere una soluzione limpida, la chiarificazione della soluzione indica la formazione di vescicole lamellari costituite dai lipidi suddetti nei rapporti selezionati dall’operatore, che saranno utilizzate per rivestire il nucleo formato al punto b. At this point, the lipid solution obtained in step e. it will be sonicated until a clear solution is obtained, the clarification of the solution indicates the formation of lamellar vesicles consisting of the aforementioned lipids in the ratios selected by the operator, which will be used to coat the nucleus formed in point b.
Potrà essere utilizzato qualsiasi sonicatore idoneo convenzionalmente utilizzato dal tecnico del settore, come ad esempio un sonicatore con punta al titanio per un periodo di circa 10 minuti. Il tecnico del settore saprà scegliere la sequenza di cicli on –off più efficace senza bisogno di ulteriori insegnamenti. A titolo meramente esemplificativo e non limitativo potrà essere utilizzata una sequenza di cicli 8 s on - 6 s off per t=10 min. Le vescicole così preparate potranno essere conservate a circa 4°C per un periodo fino a 1 o 2 settimane, e potranno essere poi direttamente utilizzate per il passaggio f. del procedimento di preparazione delle nanoparticelle dell’invenzione. Any suitable sonicator conventionally used by the person skilled in the art, such as a titanium tipped sonicator, can be used for a period of approximately 10 minutes. The technician in the sector will be able to choose the most effective on-off cycle sequence without the need for further training. By way of non-limiting example, a sequence of 8 s on - 6 s off cycles for t = 10 min can be used. The vesicles thus prepared can be stored at about 4 ° C for a period of up to 1 or 2 weeks, and can then be directly used for step f. of the process for preparing the nanoparticles of the invention.
In questo passaggio si mescola un opportuno volume di dette vescicole con un opportuno volume del complesso equilibrato ottenuto al punto b. (in una forma esemplificativa, ovviamente non limitativa dell’invenzione, si possono mescolare circa 5 microlitri di vescicole con 2 microlitri di condensante/acido nucleico) ad un rapporto di carica (moli di lipide cationico/basi di acido nucleico Ï compreso tra circa 2 e 3, estremi inclusi. Tale rapporto, in una forma esemplificativa non limitativa, potrà essere ad esempio circa 2,5. In this step, a suitable volume of said vesicles is mixed with a suitable volume of the balanced complex obtained at point b. (in an exemplary form, obviously not limiting the invention, about 5 microliters of vesicles can be mixed with 2 microliters of condensant / nucleic acid) at a charge ratio (moles of cationic lipid / bases of nucleic acid Ï between about 2 and 3, extremes included.This ratio, in a non-limiting exemplary form, could be for example about 2.5.
Il calcolo del rapporto di carica Ï può essere fatta come segue (l’esempio si riferisce ad una forma di realizzazione in cui l’acido nucleico à ̈ DNA): The calculation of the charge ratio Ï can be done as follows (the example refers to an embodiment in which the nucleic acid is DNA):
il numero di moli di lipide cationico à ̈ dato dal calcolo: the number of moles of cationic lipid is given by the calculation:
massa lipide cationico/peso molecolare del lipide cationico; cationic lipid mass / molecular weight of cationic lipid;
il numero di nucleotidi à ̈ dato dal calcolo: the number of nucleotides is given by the calculation:
massa di DNA/324.5 (peso molecolare di un nucleotide che contiene una carica negativa). mass of DNA / 324.5 (molecular weight of a nucleotide that contains a negative charge).
Da qui, rho=Numero di moli di lipide cationico/Numero di nucleotidi (o di basi). Hence, rho = Number of moles of cationic lipid / Number of nucleotides (or bases).
L’incubazione potrà essere effettuata per un periodo compreso tra circa 30 minuti e 3 ore, ad esempio un periodo di 30, 40, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 minuti circa. The incubation can be carried out for a period between about 30 minutes and 3 hours, for example a period of 30, 40, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 minutes approximately.
In una forma di realizzazione molto efficace sono preparate nanoparticelle multicomponente in cui il nucleo à ̈ formato da acido nucleico e protammina in rapporto di volume acido nucleico/protammina pari a circa 1:0,5 partendo da soluzioni iniziali aventi una concentrazione c=1 mg/ml. Il nucleo si presenta dimensioni con diametro D=230±18 nm e carica superficiale =-19,5 ± 2,5 mV. In a very effective embodiment, multicomponent nanoparticles are prepared in which the nucleus is formed by nucleic acid and protamine in a nucleic acid / protamine volume ratio equal to about 1: 0.5 starting from initial solutions having a concentration c = 1 mg / ml. The core has dimensions with diameter D = 230 ± 18 nm and surface charge = -19.5 ± 2.5 mV.
Le soluzioni di DOTAP, DC-Chol, DOPE e DOPC sono preparate in cloroformio ad una frazione molare di lipide neutro nel doppio strato Ф = (lipide neutro/lipide totale) (mol/mol) pari a 0,5 e il rapporto reciproco in cui sono miscelate al punto c. (nell’ordine sopra indicato) à ̈ scelto tra The solutions of DOTAP, DC-Chol, DOPE and DOPC are prepared in chloroform to a mole fraction of neutral lipid in the bilayer Ф = (neutral lipid / total lipid) (mol / mol) equal to 0.5 and the reciprocal ratio in which they are mixed in step c. (in the order indicated above) is chosen from
• DOTAP-DC-Chol-DOPC-DOPE (1.5:0.5:1.5:0.5) â € ¢ DOTAP-DC-Chol-DOPC-DOPE (1.5: 0.5: 1.5: 0.5)
• DOTAP-DC-Chol-DOPC-DOPE (1:1:1:1) â € ¢ DOTAP-DC-Chol-DOPC-DOPE (1: 1: 1: 1)
• DOTAP-DC-Chol-DOPC-DOPE (0.5:1.5:0.5:1.5). â € ¢ DOTAP-DC-Chol-DOPC-DOPE (0.5: 1.5: 0.5: 1.5).
La soluzione contenente il nucleo e quella contenente le vescicole multicomponente ottenute come sopra descritto sono miscelate in modo tale che il rapporto di carica (mole di lipide cationico/base di acido nucleico sia pari a circa 2,5. The solution containing the nucleus and that containing the multicomponent vesicles obtained as described above are mixed in such a way that the charge ratio (mole of cationic lipid / nucleic acid base is equal to about 2.5.
Le nanoparticelle ottenute presentano la composizione in lipidi nel rivestimento con rapporti reciproci pari a quelli utilizzati nel passaggio di miscelazione c. The obtained nanoparticles have the lipid composition in the coating with reciprocal ratios equal to those used in the mixing step c.
Sono ovviamente anche oggetto dell’invenzione le nanoparticelle definite come “nanoparticelle ottenibili mediante il procedimento dell’invenzione come definito nella descrizione e nelle rivendicazioni relative detto procedimento†. The nanoparticles defined as â € œnanoparticles obtainable by means of the process of the invention as defined in the description and in the relative claims of said processâ € are obviously also object of the invention.
Come già indicato, sono oggetto della presente invenzione le nanoparticelle come qui descritte per uso nella terapia genica. As already indicated, the object of the present invention is the nanoparticles as described herein for use in gene therapy.
In altri termini, à ̈ oggetto dell’invenzione un metodo di terapia genica che comprende il passaggio di somministrare ad un paziente che ne abbia bisogno, le nanoparticelle dell’invenzione. In other words, the object of the invention is a method of gene therapy which includes the step of administering the nanoparticles of the invention to a patient who needs it.
E’ evidente che le nanoparticelle saranno realizzate con gli opportuni acidi nucleici selezionati in relazione alla terapia genica desiderata. It is evident that the nanoparticles will be made with the appropriate nucleic acids selected in relation to the desired gene therapy.
Senza legarsi a specifiche teorie, gli inventori della presente invenzione hanno formulato le seguenti ipotesi. Without binding to specific theories, the inventors of the present invention have formulated the following hypotheses.
Le interazioni elettrostatiche non sono sufficienti a descrivere la formazione e la stabilità termodinamica del nucleo DNA/agente condensante, ma à ̈ necessario riferirsi al meccanismo del rilascio dei contro-ioni. La condensazione di Poisson-Boltzman prevede che le macromolecole cariche siano circondate in soluzione da uno strato diffuso di contro-ioni. Sotto formazione del complesso, le macromolecole rilasciano i propri contro-ioni in soluzione con un notevole guadagno entropico del sistema(circa 1 KT per ogni contro-ione rilasciato). Lo stesso meccanismo d’azione à ̈ in grado di giustificare la formazione della nanoparticella. L’aspetto più significativo à ̈ la straordinaria capacità esibita dalle nanoparticelle lipidiche a multicomponenti di rilasciare il DNA nel citoplasma e nel nucleo della cellula (vedi Figura 3). Questa proprietà rende uniche le nanoparticelle lipidiche a multicomponenti ed à ̈ la spiegazione più probabile della loro elevata efficienza (Figura 1). Questa intrinseca capacità di uscire dagli endosomi e rilasciare il DNA à ̈ probabilmente dovuta, tra gli altri fattori, al numero delle specie lipidiche coinvolte che sono in grado di massimizzare l’entropia di mescolamento lipidico (lipid mixing) rendendo energeticamente favorito il mescolamento con le specie lipidiche delle membrane endosomiali (Caracciolo et al. The Journal of Physical Chemistry B 110, 20829-20835 (2006)). Questo mescolamento sembra essere necessario per la formazione degli intermedi strutturali che conducono alla fusione del doppio strato lipidico della nanoparticelle e quello delle membrane degli endosomi. In più, il ridotto numero di strati del guscio lipidico rispetto ai lipoplessi di stessa composizione à ̈ compatibile con un più semplice disassemblaggio delle nanoparticelle rispetto ai lipoplessi multilamellari. The electrostatic interactions are not sufficient to describe the formation and thermodynamic stability of the DNA / condensing agent nucleus, but it is necessary to refer to the mechanism of the release of the counter-ions. The Poisson-Boltzman condensation predicts that charged macromolecules are surrounded in solution by a diffuse layer of counter-ions. Under the formation of the complex, the macromolecules release their counter-ions in solution with a considerable entropic gain of the system (about 1 KT for each counter-ion released). The same mechanism of action is able to justify the formation of the nanoparticle. The most significant aspect is the extraordinary ability exhibited by the multicomponent lipid nanoparticles to release DNA into the cytoplasm and nucleus of the cell (see Figure 3). This property makes multicomponent lipid nanoparticles unique and is the most likely explanation for their high efficiency (Figure 1). This intrinsic ability to exit the endosomes and release DNA is probably due, among other factors, to the number of lipid species involved which are able to maximize the entropy of lipid mixing, making it energetically favored mixing with lipid species of endosomal membranes (Caracciolo et al. The Journal of Physical Chemistry B 110, 20829-20835 (2006)). This mixing seems to be necessary for the formation of the structural intermediates that lead to the fusion of the lipid bilayer of the nanoparticles and that of the membranes of the endosomes. In addition, the reduced number of layers of the lipid shell compared to the lipoplexes of the same composition is compatible with a simpler disassembly of the nanoparticles compared to the multilamellar lipoplexes.
I seguenti esempi hanno lo scopo di illustrare, senza tuttavia limitarla al loro contenuto, l’invenzione in modo da fornire al tecnico del settore esempi concreti di realizzazione dell’invenzione. The following examples have the purpose of illustrating, without however limiting it to their content, the invention in order to provide the skilled person with concrete examples of realization of the invention.
ESEMPI e PARTE SPERIMENTALE EXAMPLES and EXPERIMENTAL PART
Gli esperimenti di trasfezione sotto riportati sono stati realizzati su linee cellulari commerciali e The transfection experiments reported below were performed on commercial cell lines and
NIH 3T3 Numero ATCC CRL-1658 NIH 3T3 ATCC number CRL-1658
HeLa Numero ATCC CCL-2 HeLa ATCC CCL-2 number
CHO (Numero ATCC CCL-61) CHO (ATCC CCL-61 number)
Su cellule murine qui denominate A17 isolate dagli inventori. On mouse cells here named A17 isolated by the inventors.
1. PROCEDURA DI PREPARAZIONE DELLE NANOPARTICELLE LIPIDICHE 1. PROCEDURE OF PREPARATION OF LIPID NANOPARTICLES
Le nanoparticelle sono costituite da un nucleo di DNA/protamina ottenuto mescolando due soluzioni di DNA e protamina in rapporto di volume DNA/protamina 1:0.75 (le soluzioni di partenza di DNA e portamina hanno la stessa concentrazione, c=1 mg/ml). Il guscio DNA/protamina à ̈ lasciato equilibrare per un'ora a temperatura ambiente. Al termine di questa procedura il guscio DNA/protamina à ̈ carico negativamente (Potenziale Zeta = -19.5 ± 2.5 mV) ed ha dimensioni caratteristiche pari circa a 230 nm (diametro idrodinamico, D = 230 ± 18 nm). Un volume complesso DNA/protamina così preparato à ̈ fatto interagire per 20 minuti con una soluzione di liposomi cationici costituiti da una miscela del lipide cationico commerciale DOTAP, del derivato cationico del colesterolo DC-Chol e da due fosfolipidi neutri come DOPE e DOPC. Le quattro specie molecolari sono mescolate in rapporto 0.5:1.5:0.5:1.5. Dopo 2 ore di incubazione, la formazione delle nanoparticelle à ̈ completa. La carica superficiale à ̈ positiva con il nucleo di DNA/protamina à ̈ ricoperto da un guscio lipidico costituito da una ad alcuni doppi-strati lipidici. La formulazione lipidica utilizzata nella presente invenzione non à ̈ mai stata precedentemente impiegata per la produzione di nanoparticelle con un nucleo di DNA/protamina. The nanoparticles consist of a DNA / protamine nucleus obtained by mixing two solutions of DNA and protamine in a DNA / protamine volume ratio 1: 0.75 (the starting solutions of DNA and portamine have the same concentration, c = 1 mg / ml) . The DNA / protamine shell is allowed to equilibrate for one hour at room temperature. At the end of this procedure, the DNA / protamine shell is negatively charged (Zeta potential = -19.5 ± 2.5 mV) and has a characteristic size of approximately 230 nm (hydrodynamic diameter, D = 230 ± 18 nm). A volume DNA / protamine complex thus prepared is made to interact for 20 minutes with a solution of cationic liposomes consisting of a mixture of the commercial cationic lipid DOTAP, the cationic derivative of cholesterol DC-Chol and two neutral phospholipids such as DOPE and DOPC. The four molecular species are mixed in the ratio 0.5: 1.5: 0.5: 1.5. After 2 hours of incubation, the formation of the nanoparticles is complete. The surface charge is positive with the DNA / protamine core covered by a lipid shell consisting of one to a few lipid bilayers. The lipid formulation used in the present invention has never previously been used for the production of nanoparticles with a DNA / protamine core.
2. PROCEDIMENTO DETTAGLIATO DI PREPARAZIONE DELLE NANOPARTICELLE UTILIZZATE NEGLI ESPERIMENTI RIPORTATI NELLE FIGURE 2. DETAILED PROCEDURE FOR PREPARING THE NANOPARTICLES USED IN THE EXPERIMENTS SHOWN IN THE FIGURES
I lipidi cationici 1,2-dioleoil-3-trimetilammoniopropano (DOTAP) e 3ß-[N-(N’,N’-dimetilamminoetano)-carbamoil] colesterolo cloridrato (DC-Chol) e i due fosfolipidi neutri dioleoilfosfatidiletanolammina (DOPE) e dioleilfosfocoliina (DOPC) sono stati acquistati dalla Avanti Polar Lipids (Alabaster, AL) ed utilizzati senza ulteriori purificazioni. Per le misure di microscopia di fluorescenza sono stati utilizzati i lipidi neutri fluorescenti DOPC-NBD e DOPE-NBD acquistati dalla Avanti Polar Lipids. I liposomi cationici a multicomponente costituiti dalle quattro specie lipidiche sono stati preparati ad una frazione molare di lipide neutro nel doppio strato Φ = (lipide neutro /lipide totale) (mol/mol) = 0.5. I lipidi sono stati sciolti in cloroformio e mescolati in modo da ottenere un miscelamento omogeneo delle quattro specie lipidiche. La quantità di cloroformio da utilizzare dipende dalla solubilità delle specie lipidiche utilizzate, in genere le soluzioni sono state preparate a circa 10/20 mg lipide/ml cloroformio. Il solvente organico à ̈ stato rimosso utilizzando un evaporatore rotante che ha portato alla formazione di un sottile film lipidico sul fondo di un’ampolla. Per assicurare la completa rimozione del solvente organico, il film lipidico à ̈ stato tenuto per 24 ore sotto vuoto. Successivamente il film lipidico à ̈ stato idratato aggiungendo il volume di tampone Tris-HCl (10 mM, pH 7.4) necessario ad ottenere una concentrazione finale di 1 mg/ml. Per ottenere vescicole unilamellari, le soluzioni lipidiche sono state sonicate utilizzando un sonicatore a punta al titanio con una sequenza di cicli (8 s on - 6 s off) per t=10 min. Le soluzioni lipidiche utilizzate sono state preparate facendo variare il rapporto tra le specie lipidiche cationiche: The cationic lipids 1,2-dioleoyl-3-trimethylammoniumopropane (DOTAP) and 3ß- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and the two neutral phospholipids dioleoylphosphatidylethanolamine and dioleylphosphocoliin (DOPC) were purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification. For fluorescence microscopy measurements, DOPC-NBD and DOPE-NBD fluorescent neutral lipids purchased from Avanti Polar Lipids were used. The multicomponent cationic liposomes consisting of the four lipid species were prepared with a mole fraction of neutral lipid in the bilayer Φ = (neutral lipid / total lipid) (mol / mol) = 0.5. The lipids were dissolved in chloroform and mixed in order to obtain a homogeneous mixing of the four lipid species. The quantity of chloroform to be used depends on the solubility of the lipid species used, generally the solutions have been prepared at about 10/20 mg lipid / ml chloroform. The organic solvent was removed using a rotary evaporator which led to the formation of a thin lipid film on the bottom of an ampoule. To ensure complete removal of the organic solvent, the lipid film was kept under vacuum for 24 hours. Subsequently the lipid film was hydrated by adding the volume of Tris-HCl buffer (10 mM, pH 7.4) necessary to obtain a final concentration of 1 mg / ml. To obtain unilamellar vesicles, the lipid solutions were sonicated using a titanium-tipped sonicator with a sequence of cycles (8 s on - 6 s off) for t = 10 min. The lipid solutions used were prepared by varying the ratio between the cationic lipid species:
• DOTAP-DC-Chol-DOPC-DOPE (1.5:0.5:1.5:0.5) â € ¢ DOTAP-DC-Chol-DOPC-DOPE (1.5: 0.5: 1.5: 0.5)
• DOTAP-DC-Chol-DOPC-DOPE (1:1:1:1) â € ¢ DOTAP-DC-Chol-DOPC-DOPE (1: 1: 1: 1)
• DOTAP-DC-Chol-DOPC-DOPE (0.5:1.5:0.5:1.5) â € ¢ DOTAP-DC-Chol-DOPC-DOPE (0.5: 1.5: 0.5: 1.5)
Il DNA a doppio filamento di timo di vitello à ̈ stato acquistato dalla Sigma (St. Louis, MO) ed utilizzato senza ulteriori purificazioni. E’ stato sciolto in acqua distillata ad una concentrazione di 1 mg/ml e sonicato con un sonicatore a punta (t=5 min., 8 s on - 6 s off) per ottenere una soluzione omogenea in lunghezza (lunghezza del DNA dalle 500 alle 1000 coppie di basi). Il DNA lineare à ̈ stato utilizzato per tutti gli esperimenti di caratterizzazione chimico-fisica delle nanoparticelle. Double-stranded DNA from calf thyme was purchased from Sigma (St. Louis, MO) and used without further purification. It was dissolved in distilled water at a concentration of 1 mg / ml and sonicated with a sonicator tip (t = 5 min., 8 s on - 6 s off) to obtain a homogeneous solution in length (length of the DNA from the 500 to 1000 base pairs). Linear DNA was used for all the chemical-physical characterization experiments of the nanoparticles.
Per gli esperimenti di trasfezione cellulare à ̈ stato utilizzato il DNA plasmidico (pGL3 che codifica per la luciferasi) acquistato dalla Promega (Madison, WI) e sciolto in acqua bidistillata (Carlo Erba Reagenti, Milano, Italia) ad una concentrazione di 1 mg/ml. Per gli esperimenti di microscopia di fluorescenza à ̈ stato utilizzato il DNA plasmidico 2.7-kbp marcato con Cy3 (Mirus Bio Corporation, Madison, WI). For the cell transfection experiments, plasmid DNA (pGL3 encoding for luciferase) purchased from Promega (Madison, WI) and dissolved in double distilled water (Carlo Erba Reagenti, Milan, Italy) at a concentration of 1 mg / ml. Cy3-labeled 2.7-kbp plasmid DNA (Mirus Bio Corporation, Madison, WI) was used for the fluorescence microscopy experiments.
La protamina solfato di salmone (MW = 5.1 kDa), acquistata dalla Sigma, à ̈ stata sciolta in acqua distillata ad una concentrazione di 1 mg/ml. Il nucleo delle nanoparticelle à ̈ stato ottenuto mescolando il DNA (lineare, plasmidico e fluorescente) e la soluzione di protammina ad un rapporto di volume 1:0.75 al fine di ottenere un complesso carico negativamente. Un volume desiderato di DNA/protamina à ̈ lasciato equilibrare per un’ora a temperatura ambiente e successivamente à ̈ stato fatto interagire con un opportuno volume della soluzione di liposomi cationici ad un rapporto di carica Ï (mole di lipide cationico/base del DNA) = 2.5. Dopo 2 ore di incubazione, la formazione delle nanoparticelle à ̈ completa. Salmon protamine sulphate (MW = 5.1 kDa), purchased from Sigma, was dissolved in distilled water at a concentration of 1 mg / ml. The nucleus of the nanoparticles was obtained by mixing the DNA (linear, plasmid and fluorescent) and the protamine solution at a volume ratio 1: 0.75 in order to obtain a negatively charged complex. A desired volume of DNA / protamine is left to equilibrate for one hour at room temperature and subsequently it has been made to interact with an appropriate volume of the cationic liposome solution at a charge ratio Ï (mole of cationic lipid / base of DNA ) = 2.5. After 2 hours of incubation, the formation of the nanoparticles is complete.
3. Protocollo per un esperimento di trasfezione (condizioni riferite ad un pozzetto di una piastra da 24 pozzetti)* ;1. 0,5 microlitri di DNA (concentrazione 1 mg/ml) sono mescolati con 0.35 microlitri di protamina (concentrazione 1 mg/ml) e lasciati incubare per 10 minuti. ;2. Sono aggiunti 49.1 microlitri di Opti-MEM® (Life Technologies) al complesso DNA/protamina formato. ;3. 5 microlitri della soluzione di liposomi cationici sono diluiti in 45 microlitri di Opti-MEM®. ;4. 50 microlitri della soluzione DNA/protamina sono mescolati con 50 microlitri della soluzione di liposomi cationici. Le nanoparticelle così formate sono lasciate incubare per 2 ore a temperatura ambiente. ;5. 100 microlitri della soluzione di nanoparticelle sono diluiti in 400 microlitri di Opti-MEM® (volume di 500 x pozzetto) e dati ad un pozzetto di una piastra da 24 pozzetti (cellule al 70% di confluenza). ;6. Dopo 4 ore, i 500 microlitri sono rimossi dal pozzetto e à ̈ aggiunto 1 ml di terreno di coltura (DMEM) complementato con siero. ;;* In questo caso il rapporto di carica delle nanoparticelle à ̈ Ï =2.5. Per raddoppiare il rapporto di carica à ̈ necessario utilizzare 10 microlitri della soluzione di liposomi cationici (punto 3). 3. Protocol for a transfection experiment (conditions referred to one well of a 24-well plate) *; 1. 0.5 microliters of DNA (concentration 1 mg / ml) are mixed with 0.35 microliters of protamine (concentration 1 mg / ml) and allowed to incubate for 10 minutes. ;2. 49.1 microliters of Opti-MEM® (Life Technologies) are added to the formed DNA / protamine complex. ; 3. 5 microliters of the cationic liposome solution are diluted in 45 microliters of Opti-MEM®. ; 4. 50 μl of the DNA / protamine solution is mixed with 50 μl of the cationic liposome solution. The nanoparticles thus formed are left to incubate for 2 hours at room temperature. ; 5. 100 microliters of the nanoparticle solution are diluted in 400 microliters of Opti-MEM® (volume of 500 x well) and given to one well of a 24-well plate (cells at 70% confluence). ; 6. After 4 hours, the 500 microliters are removed from the well and 1 ml of culture medium (DMEM) complemented with serum is added. ;; * In this case the charge ratio of the nanoparticles is ̈ Ï = 2.5. To double the charge ratio it is necessary to use 10 microliters of the cationic liposome solution (step 3).
4. VALUTAZIONE DELL’EFFICACIA DI TRASFEZIONE (FIGURA 3) QUANTIFICAZIONE DELLATRASFEZIONE 4. ASSESSMENT OF TRANSFECTION EFFECTIVENESS (FIGURE 3) QUANTIFICATION OF TRANSFECTION
I dati relativi all’efficienza di trasfezione sono riportati in figura 1 come istogramma e nella figura 3 come immagini di fluorescenza. The data relating to the transfection efficiency are reported in figure 1 as a histogram and in figure 3 as fluorescence images.
a. Prelievo dei lisati cellulari to. Collection of cell lysates
48 ore dopo la trasfezione sono stati prelevati i lisati cellulari da utilizzare per l’analisi al luminometro e allo spettrofotometro. In ogni pozzetto della piastra di coltura: 48 hours after transfection, cell lysates were collected to be used for luminometer and spectrophotometer analysis. In each well of the culture plate:
- Ã ̈ stato aspirato il terreno ed effettuato un lavaggio con PBS a temperatura ambiente (1 ml/pozzetto); - The medium was aspirated and washed with PBS at room temperature (1 ml / well);
- sono stati aggiunti 200 microlitri di tampone di lisi †̃Lysis Buffer’ (Promega) e il lisato cellulare à ̈ stato recuperato e trasferito in una eppendorf opportunamente contrassegnata e mantenuta in ghiaccio. - 200 microliters of lysis buffer â € ̃Lysis Bufferâ € ™ (Promega) were added and the cell lysate was recovered and transferred to an appropriately marked eppendorf and kept on ice.
A questo punto ogni provetta à ̈ stata mescolata con vortex (10 secondi circa) e centrifugata (2 minuti, 12000 g, 4° C); il sovranatante à ̈ stato poi aliquotato in due nuovi tubi e conservato a – 80° C. At this point each tube was mixed with vortex (about 10 seconds) and centrifuged (2 minutes, 12000 g, 4 ° C); the supernatant was then aliquoted into two new tubes and stored at â € “80 ° C.
b. Letture al luminometro b. Luminometer readings
Per l’analisi quantitativa dell’attività della luciferasi, quale indice dell’efficienza di trasfezione, i lisati delle cellule sono stati saggiati al luminometro (Berthold AutoLumat luminometer LB-953). A tale scopo, a 20 microlitri di ciascun campione sono stati aggiunti 100 microlitri di substrato (luciferina) e subito letti per 10 secondi (2 sec di delay), seguendo il protocollo allegato al kit Promega’s Luciferase Assay System. La reazione che avviene prevede la conversione in luce dell’energia chimica liberata dall’ossidazione della luciferina in ossiluciferina, catalizzata dalla luciferasi. L’intensità di luce, rimanendo costante per almeno un minuto, à ̈ acquisita come valore numerico che corrisponde all’area sottesa alla curva di emissione nell’intervallo di tempo impostato. Ogni campione à ̈ stato misurato in doppio. Per normalizzare i valori ottenuti rispetto alle proteine totali presenti nei lisati, poi, gli stessi campioni sono stati analizzati allo spettrofotometro. For the quantitative analysis of luciferase activity, as an index of the transfection efficiency, the cell lysates were tested on the luminometer (Berthold AutoLumat luminometer LB-953). For this purpose, 100 microliters of substrate (luciferin) were added to 20 microliters of each sample and immediately read for 10 seconds (2 sec delay), following the protocol attached to the Promega's Luciferase Assay System kit. The reaction that takes place involves the conversion into light of the chemical energy released by the oxidation of luciferin into oxyluciferin, catalyzed by luciferase. The light intensity, remaining constant for at least one minute, is acquired as a numerical value which corresponds to the area under the emission curve in the set time interval. Each sample was measured in duplicate. To normalize the values obtained with respect to the total proteins present in the lysates, the same samples were then analyzed on the spectrophotometer.
Reazione bioluminescente catalizzata dalla luciferasi di lucciola (Firefly Luciferase). Bioluminescent reaction catalyzed by Firefly Luciferase.
Letture allo Spettrofotometro Spectrophotometer readings
Le letture al luminometro sono state normalizzate per i milligrammi di proteine cellulari totali presenti nei lisati, usando il Bio-Rad Protein Assay Dye Reagent (Bio-Rad), secondo il metodo di Bradford. Luminometer readings were normalized for milligrams of total cellular protein present in the lysates, using the Bio-Rad Protein Assay Dye Reagent (Bio-Rad), according to the Bradford method.
Allo scopo di ottenere dei valori di riferimento con cui confrontare i dati sperimentali, à ̈ stata costruita una curva di taratura, partendo da una soluzione madre di BSA (SieroAlbumina Bovina) a concentrazione nota (2 mg/ml) e diluendo 100 microlitri di quest’ultima in 2 ml finali di H2O. Sono state quindi saggiate due serie da 6 cuvette contenenti quantità scalari di BSA, H2O e Bio-Rad: In order to obtain reference values with which to compare the experimental data, a calibration curve was constructed, starting from a stock solution of BSA (Bovine Serum Albumin) at a known concentration (2 mg / ml) and diluting 100 microliters of this. € ™ last in 2 final ml of H2O. Two series of 6 cuvettes containing scalar quantities of BSA, H2O and Bio-Rad were then tested:
- 800 microlitri H2O 200 microlitri Bio-Rad - 800 microliters H2O 200 microliters Bio-Rad
- 20 microlitri BSA 780 microlitri H2O 200 microlitri Bio-Rad - 20 microliters BSA 780 microliters H2O 200 microliters Bio-Rad
- 50 microlitri BSA 750 microlitri H2O 200 microlitri Bio-Rad - 50 microliters BSA 750 microliters H2O 200 microliters Bio-Rad
- 100 microlitri BSA 700 microlitri H2O 200 microlitri Bio-Rad - 100 microliters BSA 700 microliters H2O 200 microliters Bio-Rad
- 150 microlitri BSA 650 microlitri H2O 200 microlitri Bio-Rad - 150 microliters BSA 650 microliters H2O 200 microliters Bio-Rad
- 200 microlitri BSA 600 microlitri H2O 200 microlitri Bio-Rad - 200 microliters BSA 600 microliters H2O 200 microliters Bio-Rad
Tracciata la curva, dunque, sono state predisposte per la lettura due cuvette per ciascun lisato di cellule trasfettate, contenenti: Plotted the curve, therefore, two cuvettes were prepared for reading for each lysate of transfected cells, containing:
- 5 microlitri di campione - 5 microliters of sample
- 795 microlitri di H2O - 795 microliters of H2O
- 200 microlitri di Bio-Rad - 200 microliters of Bio-Rad
Subito prima della lettura a 595 nm il contenuto di ogni cuvetta deve essere agitato con vortex e lasciato al buio, per un tempo compreso tra i 5 ed i 60 minuti. Immediately before reading at 595 nm, the contents of each cuvette should be vortexed and left in the dark for between 5 and 60 minutes.
Analisi dei dati Data analysis
L’analisi statistica dei dati ottenuti dalle misurazioni à ̈ stata effettuata utilizzando il programma Microsoft Excel. I valori di Efficienza di Trasfezione TE (Relative Light Unità /mg proteina) sono stati calcolati come media di almeno quattro osservazioni sperimentali. Sugli stessi campioni sono state determinate anche la deviazione standard e il S.E.M. (Standard Error of the Mean). The statistical analysis of the data obtained from the measurements was carried out using the Microsoft Excel program. TE (Relative Light Unit / mg protein) Transfection Efficiency values were calculated as the average of at least four experimental observations. The standard deviation and the S.E.M. were also determined on the same samples. (Standard Error of the Mean).
5. VALUTAZIONE DELLA CITO-TOSSICITA’ 5. ASSESSMENT OF CYTO-TOXICITY
Un istogramma dei risultati ottenuti dal saggio sotto riportato à ̈ presentato in figura 2. Le cellule vive sono in grado di ridurre chimicamente il composto MTT (un sale di tetrazolio solubile in acqua) a sale di formazano insolubile in acqua e di color violetto (Mosmann, T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods, 1983. 65(1-2): p. 55-63). Il test di tossicità delle nanoparticelle lipidiche multicomponente à ̈ stato eseguito su cellule ovariche CHO e 3T3 seminate su una piastra da 96 pozzetti (8000 cellule per pozzetto), in maniera tale da avere 16 pozzetti per condizione sperimentale. I trattamenti sono stati effettuati in 100 microlitri di Optimem finali per pozzetto da 96: 80 microlitri di Optimem 20 microlitri nanoparticelle lipidiche multicomponente (generate utilizzando 0,1 microgrammi di DNA per pozzetto e mantenendo gli stessi rapporti tra DNA e protamina e tra DNA/protamina e liposomi cationici descritti nel paragrafo 3: protocollo di trasfezione). A histogram of the results obtained from the assay reported below is presented in Figure 2. Live cells are able to chemically reduce the compound MTT (a water-soluble tetrazolium salt) to a water-insoluble, violet-colored formazan salt (Mosmann , T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods, 1983. 65 (1-2): p. 55-63). The toxicity test of multicomponent lipid nanoparticles was performed on CHO and 3T3 ovarian cells seeded on a 96-well plate (8000 cells per well), in such a way as to have 16 wells per experimental condition. The treatments were carried out in 100 microliters of final Optimem per well 96: 80 microliters of Optimem 20 microliters multicomponent lipid nanoparticles (generated using 0.1 micrograms of DNA per well and maintaining the same ratios between DNA and protamine and between DNA / protamine and cationic liposomes described in paragraph 3: transfection protocol).
Dopo incubazione con le nanoparticelle (19 ore), al mezzo di coltura à ̈ aggiunto l'MTT (concentrazione finale di 0,5 mg/ml) e la piastra à ̈ posta al buio a 37°C per 4 ore. Successivamente à ̈ eliminato dai pozzetti il terreno, aspirandolo delicatamente. Dopo essersi accertati che non siano rimaste tracce di soluzione nei pozzetti, si aggiungono 100 microlitri di DMSO a ciascuno di essi e si agita la piastra per 15 minuti a temperatura ambiente al buio allo scopo di sciogliere i cristalli del sale di formazano. Se le cellule sono vive e metabolicamente attive, effettuano la riduzione dell'MTT e quando à ̈ aggiunto il DMSO si osserva la formazione di un colore violetto. La vitalità à ̈ quantificata mediante lettura dell'assorbanza tramite un lettore di piastre ELISA, ad una lunghezza d'onda di 540 nm. I dati sono espressi come percentuale rispetto al controllo rappresentato da cellule non trattate con le miscele lipidiche. After incubation with the nanoparticles (19 hours), MTT (final concentration of 0.5 mg / ml) is added to the culture medium and the plate is placed in the dark at 37 ° C for 4 hours. Subsequently, the soil is removed from the wells, gently sucking it up. After making sure that there are no traces of solution left in the wells, 100 microliters of DMSO are added to each well and the plate is shaken for 15 minutes at room temperature in the dark to dissolve the formazan salt crystals. If the cells are alive and metabolically active, they carry out the reduction of MTT and when DMSO is added, a violet color is observed. Viability is quantified by reading absorbance with an ELISA plate reader, at a wavelength of 540 nm. The data are expressed as a percentage with respect to the control represented by cells not treated with the lipid mixtures.
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