Novel biodegradable cationic core shell nanoparticles for codelivery of drug and DNA chapter 5 conclusions and recommendations

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Novel biodegradable cationic core shell nanoparticles for codelivery of drug and DNA chapter 5 conclusions and recommendations

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Chapter Conclusions and Recommendations 5.1 Conclusions Polycations are promising non-viral vectors for gene delivery, which provide many advantages over viral vectors However, low efficiency of gene transfection provided by polycations limits their application Codelivery of various drugs with plasmid DNA has been reported to improve gene transfection and achieve synergistic effect of drug and gene therapies In this study, biodegradable cationic amphiphilic copolymers have been developed These copolymers can form core-shell structured micelles with a hydrophobic core for encapsulation of drug molecules and a cationic shell for DNA binding Theoretically, these cationic micelles can be used to carry drug and gene simultaneously For the first time, it has been proved by this study that cationic core-shell structured micelles can be used for codelivery of drug and gene Successful in vitro and in vivo gene transfection has been achieved using these cationic micelles These cationic micelles may make a promising carrier for drug and/or gene delivery 5.1.1 Biodegradable cationic amphiphilic polymers P(MDS-co-CES) and P(MDA-co-CEA) have been designed to have hydrophobic pendant groups as the core-forming block and a cationic main chain as the shell-forming block The main chain PMDS or PMDA possesses a polyester structure carrying tertiary 178 amine and quaternary ammonium It can be hydrolyzed and is used as the shell-forming segment Cholesterol is the pendant group and used as the core-forming segment The positive charges of the micelles come from the quaternary ammonium and protonization of tertiary amines in acidic solution It is believed that if the positive charges are well distributed on the surface of the micelles, they must have well-controlled particle size after complexation with DNA and strong DNA binding ability because there is no steric hindrance for DNA binding PMDS and PMDA were synthesized by condensation reaction, in which triethylamine was added to absorb hydrogen chloride produced Since the monomer methyl diethanolamine carried tertiary amine, which could also absorb hydrogen chloride Therefore, an excessive amount of triethylamine needed to be added to compete with the monomer and the main chain of the produced polymer The protonization of the tertiary amine by hydrogen chloride might not affect this reaction but it would definitely affect grafting of cholesterol, which was performed via quaternization reaction Removal of unreacted monomers after the synthesis of precursor polymer is also critical to the further reaction The precursor polymer was purified with either washing with or extraction with sodium chloride- saturated water The former method could produce PMDS or PMDA with high molecule weight but was not efficient The latter one provided very pure PMDS and PMDA For the synthesis of PEG-grafted P(MDS-co-CES) or PEG-grafted (PMDA-co-CEA), monomethyl poly(ethyl glycol) was conjugated onto the main chain as terminating agent of the condensation reaction The cholesterol group (Be-chol) was grafted onto PMDS, PMDA, PEG-PMDS and PEG-PMDA by quaternization The grafting degree of cholesterol could be varied by 179 changing the molar ratio of Be-chol to PMDS or PMDA in the quaternization reaction (See Table 4.1) In fact, the hydrophilicity of the main chain, which served as the shell of the micelles, was mainly attributed to the existence of positive charges of quaternary ammonium This is very different from commonly used cationic materials This property allowed the micelles to have DNA binding ability even in DI water On the other hand, there were tertiary amines existing in the main chain, which could be protonized in an acidic environment Therefore, to improve their DNA binding ability, the micelles were complexed with DNA in an acidic solution such as sodium acetate buffer with pH 4.6 The hydrophilicity of the polymers could be adjusted not only by the grafting degree of cholesterol but also by environmental pH Moreover, primary and secondary amines can also be incorporated into the main chain for improvement of hydrophilicity and endosome escaping ability These adjustments could actually affect the stability, size, zeta potential and ultimately gene transfection of the micelles 5.1.2 Cationic micelles The cationic amphiphilic copolymers P(MDS-co-CES) and P(MDA-co-CEA) could self-assemble into core-shell structured micelles in DI water and sodium acetate buffer with pH of 5.6 and 4.6 at very low concentrations For example, the CMC of P(MDS-coCES) (Batch No 120902b), obtained from the I338/I333 ratios, was 1.9, 1.9 and 1.5 mg/L in DI water and 0.02M sodium acetate buffer with pH of 5.6 and 4.6 respectively The CMC of P(MDA-co-CEA) (Batch No 111002b) in DI water, obtained from the I338/I333 ratios, was 2.4 mg/L After PEG conjugation, the CMC increased slightly For example, the CMC of PEG5000-P(MDS-co-CES), obtained from the I338/I333 ratios was 13.2, 18.8, 180 13.2 mg/L in DI water and 0.02M sodium acetate buffer with pH of 5.6 and 4.6 respectively The molecular weight of PEG used could be optimized to yield micelles with stable core-shell structure at low concentrations These results suggest that the micelles could form under neutral and acidic conditions, and they had a very stable coreshell structure because of the rigid chemical structure of the core-forming segment cholesterol The TEM scans of the micelles also evidenced the formation of the nanoparticles The stability of the micelles in DI water, acidic buffer, PBS buffer and PBS buffer containing serum or BSA of different concentrations was characterized by particle size The buffer concentration (ionic strength) and pH were the two main factors to influence the size of the micelles At lower pH and ionic strength, the particle size was smaller The micelles remained stable in DI water for more than one month However the size of the micelles in PBS buffer tended to increase slightly as a function of time because of the neutralization of the positive charges of the micelles by anions existing in the buffer The presence of proteins in the blood affected the stability of the micelles An increased protein concentration yielded greater size of the micelles because of the adsorption of proteins on the surface In PBS buffer containing 10% serum, particle size increased immediately up to 425 nm from about 255 nm In the presence of 1% and 3% BSA, the size of the micelles changed in a narrow range from 135 to 161 nm This may be due to the presence of small BSA particles having a size of about 10 to 20 nm However, the conjugation of PEG could improve the stability of the micelles, which were designed for systemic delivery of drug and/or gene 181 5.1.3 Encapsulation of drug molecules Two model drug compounds, indomethacin and pyrene, were encapsulated into P(MDS-co-CES) and P(MDA-co-CEA) micelles by dialysis in DI water, 0.02M sodium acetate buffer with pH of 5.6 and 4.6 P(MDA-co-CEA) micelles provided greater loading capacity probably due to the presence of a larger amount of small micelles After drug loading, the particle size increased while the zeta potential decreased An increased drug loading level led to bigger micelles A reduced pH value resulted in greater encapsulation efficiency of both pyrene and indomethacin Compared to indomethacin, pyrene had a more rigid structure and might need more time to interact with the coreforming segment cholesterol and assemble into the micelles, resulting in lower encapsulation efficiency However, at lower pH, the hydrophilicity and hydrophobicity of the polymer might be well balanced, providing more time for pyrene to assemble into the core of the micelles during the dialysis process This improved the loading efficiency of pyrene Moreover, the weight ratio of polymer to drug also affected the loading efficiency of drugs An increased polymer/drug weight ratio yielded greater encapsulation efficiency and slight improvement of drug loading level An increased drug loading level led to bigger micelles More importantly, after drug loading, size distribution became much more uniform and narrow possibility because of the interaction between the drug and the core-forming segment cholesterol The drug-loaded micelles had suitable size for in vivo application and high zeta potential for DNA binding 5.1.4 DNA binding ability 182 The DNA binding ability of P(MDS-co-CES), P(MDA-co-CEA) and PEG5000P(MDS-co-CES) micelles were studied at different pH and buffer concentration by competition binding assays and agarose gel electrophoresis The DNA binding ability of P(MDS-co-CES) and P(MDA-co-CEA) micelles was affected by pH and ionic strength With decreasing pH from 7.0 (DI water) to 4.6, the DNA binding ability of the micelles increased because more tertiary amines of the polymer were protonized and greater zeta potential was obtained Increasing ionic strength in the buffer weakened the DNA binding ability of the micelles probably because ions could compete either with DNA for the micelles or with the micelles for DNA P(MDS-co-CES) and P(MDA-co-CEA) micelles fabricated in 0.02M sodium acetate buffer with pH of 4.6 exhibited strong DNA binding ability The PEGylation of P(MDS-co-CES) reduced the DNA binding ability of the micelles In particular, PEG5000-P(MDS-co-CES) micelles showed much lower DNA binding ability than P(MDS-co-CES) micelles because of the shielding effect of PEG Using PEG of low molecular weight is expected to improve their DNA binding ability Indomethacin- and pyrene-loaded P(MDS-co-CES) micelles also exhibited strong DNA binding ability Compared to P(MDS-co-CES) micelles, the DNA binding ability of the drug-loaded P(MDS-co-CES) micelles was slightly lower because of the reduced zeta potential and the increased particle size after drug loading The complete retardation of DNA was achieved at the N/P ratio of about and for the blank P(MDS-co-CES) micelles and the indomethacin- or pyrene-loaded P(MDS-co-CES) micelles respectively The size of the micelles/DNA complexes was slightly bigger than the blank micelles The complexes were stable in aqueous solution even at the zero zeta potential point when 183 they were prepared by adding the micelles into DNA solution However, when DNA was added into the micelle solution, the micelles tendered to aggregate at the zero zeta potential point 5.1.5 Structural integrity of drug-loaded micelles during the DNA binding process The structural integrity of drug-loaded micelles during the DNA binding process and in the growth medium with 10% FBS were evaluated by using pyrene as a probe When pyrene partitioned into the core of P(MDS-co-CES) micelles from an aqueous solution, the I338/I333 ratio increased, indicating that the core of the micelles was more hydrophobic than the aqueous solution With DNA binding at the N/P ratio of 0.2 to 6, the I338/I333 ratio further increased, suggesting that the microenvironment of pyrene was more hydrophobic after DNA binding DNA molecules bound on the surface of nanoparticles enhanced the stability of core-shell structure On the other hand, the size of the micelles ranged from 152 to 299 nm at the N/P ratio of 0.4 to 10, indicating that the micelles did not collapse during the process of DNA binding Furthermore, the growth medium has also been evidenced to further enhance the structural integrity of the micelles This indicates that unlike other cationic polymer/DNA complexes the complexes can maintain the structural integrity with the presence of serum This is significant for the gene transfection, 5.1.6 In vitro and in vivo gene expression In vitro gene transfection was performed in various cell lines and primary human dermal fibroblasts using P(MDS-co-CES), PMDS and pegylated P(MDS-co-CES) 184 micelles as the vector P(MDS-co-CES) micelles provided similar level of luciferase expression in HEK293 and HepG2 cells as PEI and higher transfection level than PEI in 4T1 However, in HeLa cells and human dermal fibroblasts, the luciferase expression efficiency obtained from P(MDS-co-CES) micelles/DNA complexes was lower than that from PEI/DNA complexes At the same N/P ratio, PEG2000-P(MDS-co-CES) micelles showed lower luciferase expression efficiency than P(MDS-co-CES) micelles because of their lower DNA binding ability However, increasing the N/P ratio could significantly improve the gene transfection efficiency of PEG2000-P(MDS-co-CES) micelle In contrast, PMDS provided much lower luciferase transfection efficiency compared to P(MDS-co-CES) and PEG2000-P(MDS-co-CES This proved that the core-shell structure with positive charge on the surface is crucial for the high gene transfection level of the polymer Furthermore, the results of GFP gene transfection performed in different cell lines also indicate that the high gene transfections of P(MDS-co-CES) were mainly achieved through its high cells uptake efficiency This shows that besides the buffering ability of the cationic polymer in acidic environment, the stability of the particles is also another concern to improve the gene transfection efficiency In addition, the enhancement of gene expression by cyclosporin A in KB-31-MA cells and by paclitaxel in 4T1 cells has proved that the micelles is a kind of excellent codelivery system for hydrophobic drug and nucleic acid including DNA, RNA For proof of the principle, in vivo gene transfection was performed in the cochlea of guinea pigs and subcutaneous breast cancer model established in balb/C mice using P(MDS-co-CES) micelles After 24 hours, the micelles/DNA complexes crossed the round window membrane and luciferase as well as GFP expression was observed in the 185 cochlea of guinea pigs The P(MDS-co-CES) micelles may be used as a carrier for gene delivery to the inner ear for correcting hearing loss At the same time, the luciferase gene expression in the breast cancer performed by local injection shows that PMDS-co-CES complexes had higher gene expression level than PEI complexes The experiment performed by tail vein injection shows that P(MDS-co-CES) complexes can successfully avoid entrapment by the lung and possess higher passive targeting ability than PEI complexes This further evidenced that P(MDS-co-CES) complexes is a very stable DNA carrier Furthermore, the in vivo codelivery of paclitaxel and luciferase gene to the tumor model via local injection also shows that the gene expression level has been improved significantly This indicates that the system is an excellent codelivery system not only in vitro but also in vivo to realize synergistic effect In conclusion, a biodegradable cationic micelle system has been designed, which can carry both drugs and DNA simultaneously These micelles exhibited a much lower cytotoxicity compared to conventionally used PEI, and yielded a comparable luciferase expression level and greater percentage of GFP-positive cells when compared to PEI Gene expression was also successfully achieved in the cochlea of guinea pigs with the micelles/DNA complexes With this unique cationic micelle system, a variety of compounds could be codelivered with plasmid DNA to enhance gene transfection and/or achieve a synergy between drug and gene therapies Such a system could be used to carry an anti-cancer drug (such as paclitaxel or doxorubicin) in its hydrophobic core, while binding a nucleic acid agent on its cationic shell The nucleic acid component might be a vector encoding an antisense molecule directed against the P-glycoprotein mRNA in the 186 target cell Such a system could inhibit P-glycoprotein expression by the target, and hence, incapacitate its ability to establish multi-drug resistance, a common trait among cancer cells This, coupled with the cytotoxic effects of the anti-cancer drug, should enhance the therapeutic effect of the system 5.2 Recommendations for future work To test PEG-grafted P(MDS-co-CES) micelles on a multi-drug resistant tumor model through systemic delivery (i.e intravenous injection) An anticancer drug, paclitaxel and si-RNA encoded plasmids can be chosen to gain the benefits that the codelivery system can provide The results should be compared with those obtained from free paclitaxel in the absence of si-RNA To conjugate biological ligands onto the shell of the micelles for active targeting One of the examples is to use folate It is well known that folate receptor is over expressed on the surface of most of the cancer cell types With the conjugated folate, active targeting of the above system, the micelles/paclitaxel/si-RNA, to tumor tissues may be realized Moreover, a chemical compound such as chloroquine that can help break down endosome membrane can be codelivered with DNA to improve gene expression in some cell lines such as human dermal fibroblasts, in which the current cationic micelles provided low gene expression efficiency Primary and secondary amines can be chemically incorporated into the main chain of the cationic polymer to improve its buffering effect, which may eventually induce 187 the breakage of endosome membrane to increase gene expression efficiency of the cationic micelles 188 ... the drug and the core- forming segment cholesterol The drug- loaded micelles had suitable size for in vivo application and high zeta potential for DNA binding 5. 1.4 DNA binding ability 182 The DNA. .. size of about 10 to 20 nm However, the conjugation of PEG could improve the stability of the micelles, which were designed for systemic delivery of drug and/ or gene 181 5. 1.3 Encapsulation of drug. .. conditions, and they had a very stable coreshell structure because of the rigid chemical structure of the core- forming segment cholesterol The TEM scans of the micelles also evidenced the formation of

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