New water-soluble oxyamino chitosans as biocompatible vectors for

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Biological and Medical Applications of Materials and Interfaces

New water-soluble oxyamino chitosans as biocompatible vectors for efficacious anticancer therapy via co-delivery of gene and drug Mohini Kamra, Parikshit Moitra, Devasena Ponnalagu, Anjali A. Karande, and Santanu Bhattacharya ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b09485 • Publication Date (Web): 22 Aug 2019 Downloaded from pubs.acs.org on August 24, 2019

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ACS Applied Materials & Interfaces

New water-soluble oxyamino chitosans as biocompatible vectors for efficacious anticancer therapy via co-delivery of gene and drug Mohini Kamra,a,b Parikshit Moitra,b Devasena Ponnalagu,c,† Anjali A. Karande,c and Santanu Bhattacharyaa,b,d,*

a

Department of Organic Chemistry, Indian Institute of Science, Bangalore 560012, Karnataka,

India. b

Technical Research Centre, Indian Association for the Cultivation of Science, Kolkata 700032,

West Bengal, India. c

Department of Biochemistry, Indian Institute of Science, Bangalore 560012, Karnataka, India.

d

School of Applied and Interdisciplinary Sciences, Indian Association for the Cultivation of

Science, Kolkata 700032, West Bengal, India.

KEYWORDS: anti-tumor, chitosan, co-delivery, drug delivery, p53, sustained gene expression

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ABSTRACT: Among the many non-viral gene delivery vectors, chitosan, being a polysaccharide of natural origin, has gained special importance. In this report, chitosan, CS has been solubilized in water by preparing its O-carboxymethyl derivative, CS(CH2COOH) with an optimum degree of carboxymethylation. This has been further derivatized to get the pyridine substituted product, (py)CS(CH2COOH) where the degree of pyridine substitution (47%) was optimised based on zeta potential measurements. The optimized formulation showed a high gene binding ability, forming nano-sized positively charged polyelectrolyte complexes with DNA. These polyplexes were stable to DNAse and physiological polyanions like heparin. They also exhibited minimal toxicity in vitro and showed transfection levels comparable to the commercial standard Lipofectamine 2000 and much higher than polyethyleneamine (MW 25 kDa). Additionally, in this study, a hitherto unknown oxyamine derivative of chitosan has been prepared by phthaloyl protection, tosylation and Gabriel’s phthalimide synthesis. Nearly 40% of the primary alcohols were successfully converted to oxyamino functionality which was used for forming oxime with the anticancer drug, doxorubicin. The pH-sensitivity of the oxime ether linkage and stability under biologically relevant conditions was then used to establish the compound as a versatile drug delivery vector. Codelivery of functional gene (pCEP4-p53) and drug (doxorubicin) was accomplished in vitro and in vivo with the chitosan-pyridine imine vector, (py)CS(CH2COOH), and the newly synthesized doxorubicin oxime ether, CS(Dox). Complete tumor regression with no tumor recurrence and appreciable survivability point to the in vivo effectiveness and biocompatibility of the designed composite formulation. Overall, the pH sensitivity of the oxime linkage aiding slow and steady drug release, together with the sustained gene expression by pyridine tethered carboxymethyl chitosan allow us to generate a nanobiocomposite with significantly high anticancer therapeutic potential.


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INTRODUCTION Although an effective and promising technique, gene therapy faces a major challenge in the area of delivery of functional nucleic acids into the target cells in their intact and active forms. Despite the high efficiency of gene delivery by various viral vectors, their medical use has potential setbacks of immunogenicity, non-specificity for target cell type, cytotoxicity, insertional mutagenesis and vector inactivation. 1 Among the various non-viral transfection techniques, cationic liposomes have proven to be quite promising due to their simplicity, efficiency and safety features.2-4 However, poor cell-specific binding and fast blood clearance often limit their applicability as gene vectors in vivo. In addition to cationic lipids, cationic polymers have emerged as another category of reliable gene transfection agents. These form stable complexes with DNA, interact with cell membranes and also offer nuclease protection to the oligonucleotide in the bloodstream. 5 Some polymers that have shown promise are chitosan, polyethylenimine (PEI), poly-L-lysine (PLL), polyamidoamine dendrimers, gelatin, etc. Among these, the efficiency of gene delivery was found to be the highest for PEI6-8 and dendrimers.9 However, being synthetic and non-biodegradable, they manifest significant cytotoxicity. On the other hand, polymers such as PLL, though biodegradable, offer poorer transfection efficiency than PEI and dendrimers.10 Chitosan is a natural polysaccharide derived from chitin that is found in the shells of crustaceans.11 It consists of 1,4-linked 2-amino-2-deoxy-D-glucopyranose units. In the league of cationic polymers, chitosan holds potential candidature as a gene carrier as it is biocompatible and lends minimal toxicity. In the human body, it is generally degraded by the lysozymes into a common amino sugar, N-acetyl glucosamine.12 It is then excreted as carbon dioxide among others after getting incorporated into the synthetic pathway of the glycoproteins. Also, it offers



various functional group sites for further modification that can help one increase its efficiency as a vector. One major hurdle in the path of the widespread use of chitosan is its poor solubility in water or in any common organic solvent. In a slightly acidic pH, the primary amines of chitosan (pKa ~ 6.5) are protonated and form polyelectrolyte complexes with the negatively charged DNA. It is known that by virtue of the formation of these polyplexes, DNA is condensed into small particles and is protected against the degradation by DNase.13 However, chitosan suffers from a major drawback of low transfection efficiency in its native state. This may be attributed to the strong DNA-chitosan binding interactions and the consequent incomplete release of DNA after endosomal escape. Many derivatives have been tailored with the aim of enhancing the rate of endosomal escape and improving the transfection efficieny of chitosan. Some recently reported examples include histidine modification at the amino group,14 and O-carboxymethylation followed by introduction of imidazole moiety at the amino group. 15 However, the efficacy of intracellular delivery was either reported to be significantly lower than the commercially available standards or such a comparison was not drawn. 14,15 With the aim of overcoming its limitation of low transfection levels, chitosan has been used in several hybrid systems in combination with other known agents for efficient gene transfer like PEI,16 gold nanoparticles,17 PLGA,18 PEG,19 etc. However, there has been only one report of a purely chitosan based system that matches the standards of commercially available gene transfection reagents. However, this system also has been shown to work only in the absence of serum.20 Targeted delivery of chitosan-DNA nanoparticles has been accomplished by attachment of RGD peptide,21 folic acid,22 mannose residues,23 transferrin and KNOB proteins.24 Although a few of them outperformed the commercial transfectant, Lipofectamine 2000, several other 4 Environment ACS Paragon Plus

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components were part of the hybrid system and the toxicity levels of the multi-component assembly became a matter of concern. As part of hybrid systems with inorganic materials, chitosan is reported to have been used for delivery of chemotherapeutic drugs. 25,26 Synergistic chemotherapy using dual drug loaded systems containing chitosan, among the many components, has also been reported.27 Simultaneously, a plethora of research is being conducted in the field of cancer therapy using co-delivery of therapeutic gene and drug.28 Physical encapsulation of doxorubicin has been achieved for the co-delivery of nucleotides in chitosan containing amphiphilic or polymeric nanoparticles.29-31 While the cytoplasmic release of drug from such encapsulated assemblies may be unregulated, the covalent linkage of drug with chitosan backbone is expected to confer a more gradual and sustained release. Various functional groups on the chitosan backbone as modification sites have been employed to enhance its transfection efficiency. Towards this end, the amine group at the C-2 is the most exploited reactive site followed by the primary hydroxyl at the C-6.32-34 However, the attachment of a bioactive group to the chitosan backbone via an oxime linkage is hitherto unknown. The synthesis of oxime ethers possesses several advantages over the conventional imine synthesis. Firstly, the reaction of an oxyamine and a carbonyl compound gives a quantitative yield of the oxime ether with no byproducts. Secondly, the formation of oxime ether is chemoselective and can tolerate a wide array of functional groups present in oligonucleotides and proteins. Most importantly, the reaction of oxime formation is not sensitive to air and moisture. The reaction is highly facile and does not need any catalyst or excessive heating to take place. 35 Owing to their stability at physiological pH, a range of oximes are found to be a component of several approved drugs.36 A selective inhibitor of c-Src kinase A has been developed using a library based on bisoxime ethers.37 These observations drive the need for incorporating this versatile oxime linkage



in the development of gene and/or drug delivery vectors. With the aim of improving delivery efficacy, the acid catalyzed hydrolysis of the oxime38 was believed to aid in the release of the therapeutic cargo under the acidic microenvironment of the late endosomes. A major reason for the limited use of oximes is the lack of availability of their oxyamine precursors. In the present study, we have successfully synthesized a stable, safe and biocompatible polymer scaffold carrying multiple oxyamine reactive sites on its surface to cater to this limitation. The oxyamine then formed an oxime ether with the ketone of doxorubicin resulting in the vector-drug conjugate CS(Dox). This report also presents a complete study of new, easy to prepare, serum compatible, highly efficient, non-toxic and biodegradable chitosan derivative that shows promise as a gene delivery vector. It has been prepared by the O-carboxymethylation of chitosan followed by the formation of the Schiff base with pyridine-4-carboxaldehyde at the primary -NH2 group, (py)CS(CH2COOH). The imine group is retained (is not reduced by borohydride) so as to confer pH sensitivity to the delivery agent. The composites of (py)CS(CH2COOH)/pDNA polyplexes with CS(Dox) were characterized and explored towards their ability to co-deliver p53 plasmid DNA and doxorubicin drug. The effect of the cell permeability and buffering capacity of (py)CS(CH2COOH) to release the therapeutic gene intracellularly was successfully combined with the pH sensitivity of the oxime ether linked chitosan to release the active drug both, in vitro and in vivo.

RESULTS AND DISCUSSION Synthesis and characterization of CS(CH2COOH). It has been shown that the solubility of chitosan near neutral pH can be enhanced by the incorporation of a carboxylic acid functionality.39 A water soluble derivative of chitosan was prepared by reaction with


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Scheme 1a. Schematic representation of synthesis of (py)CS(CH 2COOH)

a

Reagents, conditions and yields: (i) ClCH2CO2H, isopropanol, rt, 24 h, quant. yield; (ii) H2O/CH3OH, rt, 4 h, 75% yield.

monochloroacetic acid (Scheme 1). Reaction with different molar ratios of monochloroacetic acid with respect to a chitosan monomer unit, leads to different degrees of grafting of the carboxymethyl substituent. The reaction was followed by FT-IR spectroscopy. Figure S1 shows the FT-IR spectra of chitosan before and after carboxymethylation where the peak at 1733 cm-1 indicates the presence of –COOH group. 1H-NMR spectra of 1A (Figure S2), reveals that the peak for the -CH2 protons of the carboxymethyl substituent overlap with the protons of the chitosan backbone and hence the degree of carboxymethylation cannot be determined from its 1H-NMR spectrum. The same was then estimated from conductometric and pH metric titrations where the respective values were obtained from the titration curves, using equation [1] given by de Abreu et al.40 ………………………….[1]



where, DS is the degree of substitution of the carboxymethyl group; M is the average molar mass of the chitosan monomer; C is the molar concentration of NaOH; V1 and V2 are the volumes (in L) of base added at the first and second inflexion point respectively; m is the mass (in g) of CS(CH2COOH) per 100 mL of solution. From equation [1], we obtained the degrees of carboxymethylation as given in Table S1. Figure S3 shows one such set of titration curves where the changes in specific conductance and pH were monitored with increasing amounts of base added. Synthesis and characterization of (py)CS(CH2COOH). Treatment of 1A with pyridine-4carboxaldehyde (Scheme 1) afforded the Schiff base with varying degrees of substitution of pyridine. The results are tabulated in Table S2. The degree of pyridine substitution was determined from the

1

H-NMR taken as given of the respective (py)CS(CH2COOH) by

integrating the aromatic peaks and comparing it to the integration value of the peak for H-1 of a glucosamine unit. The DS was further ascertained from the integration of the imine proton. Figure 1 shows the 1H-NMR spectrum of 47% substituted product. MALDI-TOF MS showed the presence of hyperfine structure with a single constant repeating unit (monomer mass) confirming purity of the synthesized polymer (Figure S4). 41,42 The increase in average molar mass from 162 g/mol in CS to 224 g/mol in (py)CS(CH2COOH), as obtained from the increase in repeating m/z unit, also confirms the degree of grafting to be ~25% O- carboxymethylated and ~45% Npyridinylated which is in line with the values obtained from 1H-NMR. Similarly, the average molar mass of CS(CH2COOH), 185 g/mol is in concordance with the value obtained for ~25% O- carboxymethylated product. From the GPC traces (Figure S5), we observed a bimodal elution profile for CS, CS(CH2COOH) and (py)CS(CH 2COOH) that may be accounted for by the aggregation behavior of the polymer in aqueous media (where such experiments have been


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performed) and further changes in the aggregation propensity and hydrodynamic volume with various degrees of functionalization. 43 Successful derivatization is confirmed by the lowering of retention time with concomitant peak broadening. 41 This is because larger molecules are eluted faster than smaller molecules, and variable functionalization across monomers leads to higher polydispersity.44 In order to deviate minimally from the naturally occuring polymer so as to achieve maximal biocompatibility, the naturally extracted polymer of a certain mass range (190310 kDa) and 80% degree of deacetylation was used for further functionalization. It is possible that in a polymeric system with multiple graftings performed, polymeric chains of different sizes and different degrees of grafting are obtained where the 1H-NMR reflects the average degree of grafting. Synthesis and characterization of chitosan oxyamine derivative, CS(ONH 2). The oxyamine derivative of chitosan at the 6-O position was prepared by N-protection followed by tosylation and reductive amination via Gabriel’s phthalimide route (Scheme 2). All the primary amines of chitosan were phthaloyl protected, 45 and characterized by FT-IR spectroscopy (KBr) (Figure S6) from the emergence of peaks at 1776 cm-1(imide C=O), 1719 cm-1 (imide C=O) and 721 cm-1 (aromatic). Complete N-protection was confirmed by elemental analysis, considering 100% N-phathaloylation, i.e., for [C12.8H13NO5.8.H2O] calcd: C, 51.46; H, 5.69; N, 5.46 and found: C, 51.85; H, 5.43; N, 5.39. Effect of phthaloyl substitution on the crystallinity of chitosan was further determined to ascertain the absence of O-phthaloylation.46 Maintenance of crystallinity after introduction of phthaloyl groups is an evidence to the structural uniformity of the product (Figure S7). The partially O-phthaloylated product has been reported to be amorphous45,47 owing to the heterogeneity in structure and bulky nature of the phthaloyl group. The native chitosan polymer



Figure 1. 1H-NMR spectrum of (py)CS(CH2COOH) in DCl/D2O.

shows, in concurrence with earlier reports,48 majorly two crystalline peaks i.e. at 2Ɵ =10.5º and 20.1º, while the crystalline N-phthaloyl chitosan, 2A showed majorly four peaks at 2Ɵ = 7º, 12.4º, 19.2º, 26.7º. This N-phthaloyl chitosan has been utilized for several synthetic modifications to improve facility, regioselectivity and reaction yield under mild reaction conditions.49 It is believed to serve as a key intermediate for further modifications on the chitosan backbone in a controlled manner. Being one of the most effective leaving groups, the (p-tolylsulfonyl)oxy or tosyl group is used widely in carbohydrate chemistry for regioselective reaction at the primary alcohol site in contradiction to the secondary alcohols. 50 Hence, 2A was treated with excess of tosyl chloride under basic conditions in dimethylacetamide solvent to yield 6-O-tosyl-N-phthaloyl chitosan,


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Scheme 2b. Synthesis of CS(ONH2)

b

Reagents, conditions and yields: (i) Phthalic anhydride, DMF/H2O (95:5),120 ºC, 8 h, quant.

yield (ii) TsCl, pyridine, DMA (1% LiCl), rt, 17 h, 70% yield (iii) N-hydroxyphthalimide, K2CO3, DMF, 90 ºC, 24 h, quant. yield (iv) Hydrazine hydrate, DMSO, 80 ºC, 24 h, quant. yield.

2B. It has been shown that both the glucosamine and N-acetyl glucosamine units were equally susceptible to tosylation at the 6-O position. In the FT-IR spectrum (Figure S8), the appearance of a new peak at 1198 cm-1 was characteristic of the newly introduced tosyl group along with the increased number of peaks in the aromatic fingerprint region. However, the degree of tosylation,



determined from the elemental analysis as ~40%, is the limiting parameter for further substitution. The tosyl group was quantitatively substituted by the phthalimide group using in situ generated potassium phthalimide intermediate.51 The di-phthaloyl chitosan, 2C, was found to be mildly soluble in water and

1

H-NMR was performed to determine the degree of substitution.

Considering that all the primary amine groups of glucosamine are phthaloyl protected, the number of protons arising from the phthalimide substitution at the 6-O position that were contributing to the aromatic signal in 1H-NMR spectrum were calculated and nearly 40% of the primary hydroxyl groups were found to be substituted (Figure S9). Thus, all the tosylated alcohol units were quantitatively transformed to phthaloyl units that were reductively hydrolyzed to generate the free oxyamine. In stark contrast to the native chitosan, the oxyamine derivative was found to be soluble in water pointing to the increased polarity of the polymer. It was characterized by 1H-NMR, FT-IR and elemental analysis. The 1H-NMR was similar to that of the starting chitosan (Figure S10) indicating the removal of all protecting groups which was also evident from the FT-IR spectrum (Figure S11). Overall, the synthetic route followed for CS(ONH2) used conventional high yielding reactions like base mediated reaction with N-hydroxyphthalimide. By first protecting the primary amines via phthaloylation, the primary alcohols were activated by tosylation. These reactive sites were aminated while generating the highly reactive potassium phthalimide in situ as an –NH2 synthon intermediate. The conversion of chitosan to a hitherto unknown oxyamine derivative was thus accomplished by combination of some suitably designed modification steps on the chitosan backbone. Synthesis and characterization of CS(Dox) oxime ether. The newly synthesized oxyamine


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derivative, CS(ONH2) was used for the preparation of doxorubicin oxime ether via Scheme 3 using the conditions that prefer oxime formation over imine formation. 52,53 The anticancer drug, doxorubicin was supplied in equimolar concentration to the oxyamine partner to further ensure that free amine at the C-2 position of chitosan does not participate in imine formation with the ketone functionality of the doxorubicin. The newly synthesized oxime ether synthesized was characterized using 1H-NMR, FT-IR and elemental analysis. The degree of doxorubicin substitution, as determined from 1H-NMR (Figure 2) was found to be nearly 40 %, which is in concurrence with the number of phthaloyl groups mounted and consequently the number of monomers carrying the reactive oxyamine site. This further affirms that only the oxyamine moieties have participated in the reaction with the ketone. The FT-IR spectrum of CS(Dox) is shown in Figure S12. Polymer Solubility. To assess the solubility limit of the synthesized chitosan derivatives with respect to pH of the solution, UV-Vis spectral titrations were performed. As opposed to the parent chitosan, the functionalized chitosan, (py)CS(CH 2COOH), shows good water solubility

Scheme 3c. Synthesis of CS(Dox) oxime ether

c

Reagents, conditions and yields: (i) Doxorubicin.HCl, phosphate buffer pH 7.4/THF (3:2), rt, 8 h, quant. yield. 13 Environment ACS Paragon Plus


Figure 2. 1H-NMR spectrum of CS(Dox) oxime ether depicting 40% Doxorubicin substitution at the 6-ONH2 position.

over a wide pH range (Figure S13). (py)CS(CH2COOH) did not show cloud point even until pH 12 whereas the parent chitosan exhibited a cloud point at around pH 7.5. Since precipitation at any of the working conditions is not acceptable in most biomedical applications, aqueous solubility at a dynamic pH range of the human body is an important criterion for an efficient transfectant. This characteristic is found to improve upon carboxymethylation and a subsequent pyridine substitution at the polymer backbone. This indicates that such a functionalization is beneficial to the polymer’s candidature as a gene delivery agent. Determination of particle size and zeta potential. Determining the sizes and zeta potential of the polymer and polyplex nanoparticles was an important parameter to assess its delivery


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capability. The DLS and zeta potential measurement data for CS(CH 2COOH) have been summarized in Figure 3A. One observes an increase in the degree of carboxymethylation with the nanoparticle size slightly but lowering the zeta potential significantly. Since we need a substitution level just enough to achieve solubility in water, and the 8% substituted product turns out to be water insoluble, we arrive at an optimum degree of carboxymethylation of 25%. Using this 25% substituted CS(CH2COOH), the reaction with pyridine-4-carboxaldehyde was carried out where a saturation in the degree of pyridine substitution was seen at a value of 47%. The size and zeta potential values for (py)CS(CH2COOH) have been summarized in Figure 3B. We see that, as expected, an increased pyridine substitution manifests as an increase in the zeta potential and a slight increase in the nanoparticle size. Using these optimized derivatives, pDNA complexes were prepared at a high N/P ratio (to ensure complete complexation) and examined for their size and zeta potential. The results have been summarized in Table 1. A significant decrease in size upon DNA condensation indicates

Figure 3. Variation in the nanoparticle size and zeta potential with (A) % degree of carboxymethylation and (B) % degree of pyridine substitution.



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that a tight electrolyte complex is formed. It can also be seen that (py)CS(CH2COOH) was able to compact DNA into nanoparticles of size less than 100 nm, which is an advantage for a delivery system. The overall zeta potential of the DNA complex is significantly positive which points towards its ability for interaction with the cell membrane and aiding the internalization of DNA inside the cell. Thus, the 25% carboxymethylated and 47% pyridine substituted products were taken for further studies. The aggregation behavior of polymer-drug conjugate in aqueous solution was also investigated by DLS and zeta potential measurements. As shown in Table 2, the average hydrodynamic diameter of the spherical aggregates of CS(Dox) is found to be close to 200 nm. The surface charge of these aggregates is slightly positive which may be sufficient for the interaction with the negatively charged cell membranes. However, for co-delivery of a therapeutic plasmid, its surface charge was increased by forming fused aggregates with the pDNA polyplexes of the gene transfecting polymer, (py)CS(CH2COOH). The fused aggregates were found to be comparatively larger in size but were able to condense pDNA into compact polyplexes with sufficient positive

Table 1. Comparison of size and zeta potential of CS, CS(CH2COOH) and (py)CS(CH2COOH) nanoparticles before and after complexation with pDNA at pH 7.4, 37 °C.

CS CS(CH2COOH) (py)CS(CH2COOH)

Hydrodynamic diameter of the polymeric particle in solution (nm) 214 ± 4 367 ± 17 397 ± 19

Hydrodynamic diameter of the polymer/DNA complex at N/P = 20 (nm) 173 ± 3 96 ± 2 87.5 ± 0.2


Zeta potential (mV) of polymer in solution 14 ± 2 12 ± 1 22 ± 2

Zeta potential (mV) of polymer/DNA complex at N/P = 20 12 ± 3 11 ± 1 21 ± 2

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surface charge. The reported sizes were obtained when DLS was recorded in HEPES buffer, pH 7.4 (near physiological conditions). To assess the stability of the formulations, DLS in PBS (pH 7.4) over 7 days was carried out. But no significant change in average hydrodynamic diameter with time was observed indicating the stability of CS(Dox) and CS(CH2COOH)/ pEGFP-C3 polyplex (Table S3). DLS in RPMI 1640 culture medium was also recorded and the sizes were found be slightly larger although it did not change with time (Table S4). Determination of polyplex shape and morphology. To discern the nature of supramolecular structures formed upon the self-assembly of the synthesized polymers and pDNA, microscopic investigations were performed. Atomic Force Microscopy (AFM) studies clearly showed the spherical morphology of the nanoparticles formed from (py)CS(CH2COOH) in aqueous media and upon complexation with plasmid DNA, pEGFP-C3. The uniformity in size and shape at the nanometer scale, as can be seen in Figure 4A, is a contributing factor to the high transfection efficiency of this vector. The spherical shape and small size aid in the cellular internalization of the polymer/DNA complex. Morphological analysis of CS(Dox) exhibited the formation of nano-sized spherical aggregates (Figure 4B) that are mostly monodisperse. The composite

Table 2. Comparison of size and zeta potential of CS(Dox), with and without mixing with (py)CS(CH2COOH)/pDNA polyplexes at pH 7.4, 37 °C. CS(Dox)

CS(Dox) + (py)CS(CH2COOH)/pDNA

Avg. hydrodynamic diameter (nm)

212 ± 10

245 ± 5

Zeta Potential (mV)

15 ± 2

23 ± 2



Figure 4. Representative AFM image of (A) (py)CS(CH2COOH)/ pEGFP-C3 polyplex, (B) CS(Dox) and (C) composite of the two in tapping mode. Scale bar = 0.5 µm.

vesicles, however, exhibited slightly distorted spherical aggregates with comparatively higher polydispersity. Determination of degree of DNA binding. (a) Agarose Gel Electrophoresis. The ability of CS, CS(CH2COOH) and (py)CS(CH2COOH) to complex duplex plasmid DNA was examined using DNA Mobility Shift Assay (MSA). Complexed DNA migrated slower than that of naked pDNA on the agarose gel in presence of an electric field. As seen from Figure S14, CS completely retarded the motion of pDNA starting from low charge ratios whereas, CS(CH2COOH) is not able to do so even at significantly higher charge ratio. For (py)CS(CH2COOH), a polymer concentration yielding N/P of 10 and more is able to retard the motion of pDNA completely, indicating complete binding. (b) Ethidium Bromide Exclusion Assay. The degree of binding of the polymer with the DNA was also confirmed by monitoring the quenching of fluorescence of DNA-EtBr complex.54 Plots for %degree of binding with increasing stoichiometric excess of polymer (represented in terms of


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N/P) (Figure S15) depict that in the electrophoresis study, CS showed considerable degree of binding even at low N/P whereas for (py)CS(CH2 COOH), and significant binding was observed only at moderate N/P values and saturation was achieved beyond those. However, CS(CH2COOH) showed a poor binding profile, as expected. Investigation of polyplex stability. (a) DNase

resistivity.

The

integrity

of

the

DNA

after

complexation

with

(py)CS(CH2COOH) was examined using agarose gel electrophoresis (Figure S16). At N/P = 5, where there was incomplete binding of the polymer with pDNA, the free DNA was digested by DNase leaving behind the complexed DNA, seen in the well. For polyplexes where all of the DNA was complexed and not moving outside the well, no effect of DNase was observed pointing towards the DNA protection ability of the polymer. The polyplexes are thus found to be DNase stable. It may be concluded that DNA compacted with the vector remains intact and functional till it is released inside the cell for its expression. (b) Heparin resistivity. For effective pDNA delivery, apart from the polyplex being stable enough to protect the DNA against degradation by DNase, the polyplex should also be stable against dissociation by glycoaminoglycans. These polyanions are known to show competitive binding with the positively charged polyelectrolytes like chitosan. The recovery in fluorescence of the DNA-EtBr complex is a measure of the release of DNA from the polyplex upon exposure to heparin. The effect of both heparin concentration and polymer concentration on DNA release were studied. As seen in Figure 5, the polyplexes with low N/P release DNA when met with moderate heparin concentration. However, for an N/P of 50, the polyplexes are completely stable even in the presence of sufficiently high q heparin/qDNA (which is representative of the competition



between heparin and DNA towards binding to the polymer).54,55 Thus, it may be seen that a stoichiometric excess of the polymer is essential in order to confer stability to the polyplex. This complies with the known explanations that the excess polymer sequesters the competing polyanion in an affinity guided manner and the polyplex is left intact. 56 The ability of heparin to dissociate (py)CS(CH2COOH) polyplexes was also examined using gel electrophoresis. Since the heparin concentration was fixed (qheparin/qDNA = 20), the ability to release DNA fell off with increase in N/P reflecting increased polyplex stability at higher polymer/DNA charge ratios. Buffering Capacity. The buffering capacity of the native polymer, CS, was found to increase upon carboxymethylation and then decreased drastically upon further substitution with pyridine (Figure S17). The initial improvement in the buffering capacity may be attributed to the zwitterionic nature of the O-carboxymethyl chitosan. The proton absorbing ability of chitosan and the synthesized derivatives may be attributed to the presence of a series of primary and

Figure 5. (A) Fluorescence recovery of DNA-EtBr complex in the presence of increasing amounts of heparin; (B) Release of pDNA at different N/P using excess heparin qheparin/qDNA = 20. Lane 1: DNA alone; Lane 2, 4, 6: complex@N/P= 5, 10, 50 respectively + heparin; Lane 3, 5, 7: complex@N/P= 5, 10, 50 respectively. ([pEGFP] = 0.2 µg/well)


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secondary amine groups along the polymeric backbone. This confers buffering effect on endosomes and leads to the release of polyplexes from the endosomal compartments. However, the relation between the buffering capacity and the transfection efficiency has been explained in various ways in literature.57,58 The underlying point among all of these is that (a) there is no direct correlation between the gene transfer efficiency and buffering capacity of the polymer and (b) in cases where a higher buffering capacity appears to enhance the transfection ability; it does so at the cost of cellular viability.

Evaluation of pH dependent drug release. A property that has quite often been correlated with escalated levels of anticancer drug delivery is the pH-sensitivity of the drug carrier. Since a typical characteristic of solid tumor microenvironments is acidosis, several pH-responsive drug delivery vectors have been designed to achieve higher therapeutic effect. 59,60 In addition to pH cues at the organ level, drug delivery vectors need to be responsive to changes in pH at the subcellular level. The pH of early endosomes drops and reaches values less than 6 at the late endosomal stage. This has been exploited as an initiator of endosomal escape for release of the drug payload via a “proton sponge” effect. 61 Conjugation of drug molecule to a polymer backbone via a pH-labile linkage revolves around the same approach. The pH-responsive nature of the oxime ether linkage, used for decorating doxorubicin pendants on the chitosan chain, was evidenced from the drug release profiles under different pH conditions (Figure 6). Since the oxime ether linkage is hydrolyzed in acidic media, the active drug is released inside the cell in a more efficient manner. The internalization and release mechanics of the drug are thus improved to achieve the desired therapeutic effect at lower doses. The designed drug conjugate showed a zero-order drug release profile. Consistent with Peppas model, 62,63 drug release at pH 6 shows an



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initial burst release (0 – 10 h) followed by a sustained release (10 – 50 h). In case of intratumoral injections, as performed herein, the initial burst release is an important contributor to efficient tumor regression.64 Additionally, the minor drug release (

New water-soluble oxyamino chitosans as biocompatible vectors for

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