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sensitive properties of PGA, these insulin encapsulated microspheres are stable in the stomach and disaggregate in the small intestine, and the bioavailability of oral insulin administration will be improved due to DCO's permeation enhancement function. Keywords: Insulin, Oral delivery, Permeation enhancer, pH sensitive, Poly(l-glutamic acid)
Fig. 2. Pathway of SPT7pTL and visualization of PGA-α-MSH in PLLRP/(PGA-α-MSH+ SPT7pTL). The image corresponds to the overlap of light transmission and fluorescence confocal images. Red and green fluorescence correspond to areas containing Rhodamine labeled PGA-α-MSH and YOYO-1 labeled SPT7pTL, respectively. Scale bar=20 μm.
Conclusion Our data suggests that the PLLRP is a promising vector for gene therapy and hormone stimulation. PLLRP is not cytotoxic. Moreover, PLLRP could lead to efficient gene delivery and hormone stimulation followed by melanin production, simultaneously. The pathway of the DNA molecules was also visualized by confocal microscopy, and we found that SPT7pTL could enter into the cell nucleus. The entry of hormone into the cytoplasm and nuclei of cells induces melanin secretion. References [1] G.Y. Wu, C.H. Wu, Receptor-mediated in vitro gene transformation by a soluble DNA carrier systemJ. Biol. Chem. 262 (1987) 4429–4432. [2] C.M. Ward, M.L. Read, L.W. Seymour, Systemic circulation of poly(L-lysine)/DNA vectors is influenced by polycation molecular weight and type of DNA: differential circulation in mice and rats and the implications for human gene therapy, Blood 97 (2001) 2221–2229. [3] T. Merdan, J. Kopecek, T. Kissel, Prospects for cationic polymers in gene and oligonucleotide therapy against cancer, Adv. Drug Deliv. Rev. 54 (2002) 715–758. [4] A. Akinc, R. Langer, Measuring the pH environment of DNA delivered using nonviral vectors: implications for lysosomal trafficking, Biotechnol. Bioeng. 78 (2002) 503–508.
doi:10.1016/j.jconrel.2011.08.077
An efficient pH sensitive oral insulin delivery system enhanced by deoxycholic acid Li Zhao1,2, Jianxun Ding1,2, Pan He1,2, Chunsheng Xiao1,2, Zhaohui Tang1, Xiuli Zhuang1, Xuesi Chen1 1 Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China 2 Graduate University of the Chinese Academy of Sciences, Beijing 100039, China E-mail address:
[email protected] (X. Chen). Summary A permeation enhancer, positively charged deoxycholyl-hyperbranched oligoethylenimine (DCO), was prepared and used to form complexes with negatively charged insulin based on electrostatic interaction. DCO/insulin complexes can be loaded into poly (lglutamic acid) (PGA) microspheres by the oil-in-oil (O/O) emulsion and solvent evaporation technique. Because of the specific pH
Introduction Oral delivery of insulin has been considered to be the most convenient and comfortable route for insulin administration. However, the bioavailability of oral insulin is very low due to many obstacles present in the gastrointestinal tract (GIT), including the acidity, enzymatic degradation in the stomach, and the low absorption in the intestinal lumen. To overcome these barriers, many oral insulin systems with pH sensitivity, enzyme dependent release, or enhanced absorption in the GIT, have been developed. The release of insulin from pH-sensitive polymeric spheres could be triggered by the increase in pH from the stomach to the intestine. Insulin was protected by the pH-sensitive shrunken polymer in the stomach, and released in the intestine where the pH-sensitive polymer was solubilized. Deoxycholic acid, as a permeation enhancer, could greatly enhance the bioavailability of insulin after the oral administration [1,2]. Herein, we report a novel oral insulin delivery system prepared by the encapsulation of DCO/ insulin complexes in pH sensitive PGA microspheres. The PGA could protect insulin in the stomach and release it in the intestine. The positive charged DCO could form complexes with negative insulin and enhance the absorption of insulin in the intestine. Experimental methods Synthesis of DCO. DCO was prepared by a condensation reaction of deoxycholic acid with hyperbranched oligoethylenimine (OEI, Mn 600) using N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDC·HCl) as catalyst. The reaction was performed for 3 d at room temperature. The reaction solution was dialyzed against deionized water for 3 d using a dialysis bag with molecular weight cut-off of 500 Da. The final product DCO was obtained as a white solid by lyophilization. Preparation of PGA microspheres. PGA was synthesized according to our previous work [3]. PGA microspheres for oral delivery were prepared by the O/O emulsion and solvent evaporation technique. Results and discussion Positive DCO cannot make complexes with negative insulin due to the formation of micelles when the concentration exceeds its critical micellization concentration (CMC). We used a pyrene-probe-based fluorescence technique to measure the CMC of DCO (Fig. 1). The CMC of DCO is 0.038 mg/mL. DCO could form globular nanoparticles with positive surface charges (Fig. 2).The diameters of the nanoparticles will change at different pHs, as shown in Fig. 3. At a low pH of 2.0, the average size of the nanoparticles is about 585 nm. The diameter of the nanoparticles would decrease with increasing pH from 2.0 to 6.8 due to deprotonation of the amino groups in the DCO. However, if the pH value further increases, the size of the nanoparticles increases presumably because of the aggregation of the nanoparticles at higher pH. The positive DCO can be used to form complexes with negative insulin based on the electrostatic interaction at the DCO concentration below its CMC (Scheme 1). PGA has been extensively investigated for drug delivery applications due to its favorable biodegradable and biocompatible properties. In our work, DCO/insulin complexes can be loaded into PGA microspheres by the O/O emulsion and solvent evaporation technique. The typical morphology of controlled microspheres is shown in
Abstracts / Journal of Controlled Release 152 (2011) e133–e191
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0.80 0.75
I339/I333
0.70 0.65 0.60 0.55 0.50 -5
-4
-3
-2
-1
0
LogC(mg/mL) Fig. 1. The intensity ratio (I339/I333) as a function of DCO concentration. Fig. 4. Typical SEM image of PGA microspheres.
Conclusion In summary, DCO has been synthesized and can self-assemble in water to form nanoparticles with a size of around 77 nm at pH 6.8. Below the CMC 0.038 mg/mL, DCO could make complexes with insulin easily, and these complexes would be loaded in PGA microspheres. The pH sensitive oral insulin delivery system enhanced by deoxycholic acid may have potential applications. Acknowledgments We are grateful for the financial support from the National Natural Science Foundation of China (20904053, 50873102, 50733003), Ministry of Science and Technology of China (2009AA03Z308), the Knowledge Innovation Program of the Chinese Academy of Sciences (KJCX2-YW-H19), and the Program of Scientific Development of Jilin Province (20090135, 20096018). References Fig. 2. Typical TEM image of DCO nanoparticles at pH 6.8.
Fig. 4. This oral delivery system is promising in oral insulin delivery application because of many advantages. Firstly, the bioactivity of insulin loaded in PGA microspheres would be protected. Secondly, the pH sensitive PGA microspheres can control the release of insulin. Thirdly, deoxycholic acid can open the tight junctions between intestinal epithelial cells; therefore the absorption of insulin in the small intestine can be enhanced. The preparation approaches and the pH sensitive drug release mechanism are shown in Scheme 1.
[1] S.K. Kim, S. Lee, S. Jin, H.T. Moon, O.C. Jeon, D.Y. Lee, Y. Byun, Diabetes correction in pancreatectomized canines by orally absorbable insulin-deoxycholate complex, Mol. Pharm. 7 (2010) 708–717. [2] R.M. Samstein, K. Perica, F. Balderrama, M. Look, T.M. Fahmy, The use of deoxycholic acid to enhance the oral bioavailability of biodegradable nanoparticles, Biomaterials 29 (2008) 703–708. [3] C.L. He, C.W. Zhao, X.H. Guo, Z.J. Guo, X.S. Chen, X.L. Zhuang, S.Y. Liu, X.B. Jing, Novel temperature- and ph-responsive graft copolymers composed of poly(l-glutamic acid) and poly(n-isopropylacrylamide), J. Polym. Sci. Part A Polym. Chem. 46 (2008) 4140–4150.
pH 2.0 pH 5.0 pH 6.8 pH 9.0 pH 12.0
10
100
1000
Rh(nm) Fig. 3. Hydrodynamic radius (Rh) of DCO aqueous solution (0.2 mg/mL) at different pHs.
Scheme 1. Illustration of insulin-loaded nanoparticle formation and pH sensitive release of insulin.
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doi:10.1016/j.jconrel.2011.08.078
Poly(ethylene oxide) grafted with low molecular weight polyethylenimines for non-viral gene transfer Meng Zheng1, Lei Zhou1, Ru Cheng1, Fenghua Meng1, Rui Peng2, Zhiyuan Zhong1 1 Biomedical Polymers Laboratory and Jiangsu Key Laboratory of Organic Chemistry, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, China 2 Institute of Functional Nano & Soft Materials (FUNSOM), Soochow University, Suzhou 215123, China E-mail address:
[email protected] (Z. Zhong). Summary A novel gene delivery system based on poly(ethylene oxide) (PEO) grafted with 1.8 kDa branched polyethylenimine (PEI) (PEO-g-PEI) was developed. Three PEO-g-PEI copolymers, i.e. PEO(13k)-g-PEI10, PEO(24k)-g-PEI10, and PEO(13k)-g-PEI22 were synthesized. All copolymers were able to condense DNA into small-sized polyplexes (b200 nm) at and above an N/P ratio of 10. The polyplexes of PEO-gPEI revealed enhanced stability in 150 mM NaCl. Interestingly, polyplexes of PEO(13k)-g-PEI10 while maintaining a low cytotoxicity showed remarkably higher transfection activities in 293 T cells as compared to the 25 kDa PEI control. Keywords: Polyethylenimine, Poly(ethylene oxide), Graft copolymers, Gene delivery Introduction Polyethylenimine (PEI) is one of the most effective non-viral gene carriers [1]. The transfection efficiency of PEI, along with their cytotoxicity, strongly depends on their molecular weights [2]. PEI with a relatively high molecular weight (e.g. 25 kDa) though showing high transfection efficiency is toxic to cells. In contrast, PEI with a relatively low molecular weight (e.g. 1.8 kDa) though having low cytotoxicity displays minimal transfection activity. In the past years, efforts have been directed to improve the transfection efficiency of low molecular weight PEI while maintaining its low cytotoxicity. For example, the transfection efficiency of low molecular weight PEI could be improved by cross-linking of PEI [3]. For in vivo studies, PEI has been modified with hydrophilic and biocompatible poly(ethylene glycol) (PEG) that could enhance the biocompatibility, improve colloidal stability and reduce cytotoxicity [4]. In this study, 1.8 kDa PEI was grafted onto PEO to obtain a series of graft copolymers for non-toxic and efficient gene delivery. The buffer capacity, DNA condensation, cytotoxicity, and transfection activity of PEO-g-PEI copolymers were investigated. Experimental methods Polymer synthesis. PEO containing allyl functional groups (PEO-gallyl) was prepared by anionic ring-opening copolymerization of allyl glycidyl ether (AGE) and ethylene oxide (EO) using diethylene glycol as an initiator (Scheme 1). PEO-g-OH was synthesized by Michael-type addition between PEO-g-allyl and mercaptoethanol under a nitrogen atmosphere at 70 °C for 2 days in the presence of AIBN. The hydroxyl groups of PEO-g-OH were activated by 4-nitrophenyl chloroformate (4-NC) which was added dropwise to a solution of 1.8 kDa PEI (PEI/ 4-NC = 8/1 mol/mol) in CH2Cl2. The resulting polymer PEO-g-PEI was purified by dialysis against deionized water. In vitro transfection and cell viability assay. The cytotoxicity and transfection activity of PEO-g-PEI copolymers were evaluated using plasmid DNA pEGFP as a reporter gene and 293 T cells in the presence of 10% serum.
Scheme 1. Synthesis of PEO-g-PEI graft copolymers.
Results and discussion Three PEO-g-PEI graft copolymers were synthesized (Table 1). 1 The number of grafted PEI chains was determined based on H NMR analysis. The acid–base titration results showed that these grafted copolymers had similar buffer capacity as 25 kDa PEI (16.2%). The biophysical characterizations revealed that PEO-g-PEI was able to effectively bind DNA and condense DNA into compact nanoparticles (b200 nm) at and above an N/P ratio of 10. PEO-g-PEI/DNA complexes showed enhanced colloidal stability in 150 mM NaCl as compared to 25 kDa PEI/DNA complexes. MTT results displayed that the cytotoxicity of PEO-g-PEI/DNA complexes was in general lower than that for 25 kDa PEI (Fig. 1). A higher molecular weight of PEO and lower number of grafted PEI chains yielded lower cytotoxicity. The polyplexes of all three
Table 1 Characteristics of PEO-g-PEI copolymers. Entry
Graft copolymersa
Mn/kDab
PEI (wt.%)b
Buffer capacityc (%)
1 2 3
PEO(13k)-g-PEI10 PEO(24k)-g-PEI10 PEO(13k)-g-PEI22
13.1–18.0 24.0–18.0 13.0–39.6
55.2 41.6 71.4
14.7 15.5 16.4
a PEO(x)-g-PEIy wherein x represents molecular weight of PEO and y the number of grafted 1.8 kDa PEI chains. 1 b Determined by H NMR spectroscopy. c Defined as the percentage of amine groups becoming protonated from pH 5.1 to 7.4.
copolymers had low toxicity at and below an N/P ratio of 20/1. The transfection studies in 293 T cells in the presence of 10% serum showed that PEO(13k)-g-PEI10 had the highest transfection activity, affording a transfection efficiency more than 4 fold higher than 25 kDa PEI at its optimal N/P ratio of 10/1 (Fig. 2). The polyplexes of PEG(24k)-g-PEI10 and PEG(13k)-g-PEI22 showed comparable transfection to 25 kDa PEI. Conclusion We have demonstrated that PEO-g-PEI graft copolymers have good solubility, high buffer capacity, and enhanced DNA condensation. The polyplexes of PEO-g-PEI show excellent colloidal stability in 150 mM NaCl and lower cytotoxicity than those of 25 kDa PEI. Significantly, polyplexes of PEO(13k)-g-PEI10 display over 4-fold higher transfection efficiency as compared to 25 kDa PEI polyplexes in 293 T cells at optimal conditions. These PEO-g-PEI copolymers may be developed for safe and efficient delivery of DNA in vivo. Acknowledgments This work is supported by National Natural Science Foundation of China (50703028, 20974073, 50973078 and 20874070).
