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ABSTRACT: Polyplex formation (complexation) and gene release from the polyplexes (decomplexation) are major events in polymeric gene delivery, however the effect of the decomplexation rate on transfection has been rarely investigated. This study employed mixed polymers of poly(L-lysine) (PLL: MW ~7.4 kDa) and reducible PLL (RPLL) (MW ~6.7 kDa) to design decomplexation rate-controllable PLL100-xRPLLx/pDNA complexes (PRLx polyplexes). The transfection efficiency of a model gene (luciferase) in MCF7 and HEK293 cell lines increased with increasing x (RPLL content) in the PRLx polyplexes until peaking at x=2.5 and x=10, respectively, after which point transfection efficiency declined rapidly. In MCF7 cells, PRL2.5 polyplex produced 3- or 223-fold higher gene expression than PLL or RPLL polyplexes, respectively. Similarly, the transfection efficiency of PRL10 polyplex-transfected HEK293 cells was 3.8- or 67-fold higher than that of PLL or RPLL polyplexes, respectively. The transfection results were not apparently related to the particle size, surface charge, complexation/compactness, cellular uptake, or cytotoxicity of the tested polyplexes. However, the decomplexation rate varied by RPLL content in the polyplexes, which in turn influenced the gene transfection. The nuclear localization of pDNA delivered by PRLx polyplexes showed a similar trend to their transfection efficiencies. This study suggests that an optimum decomplexation rate may result in high nuclear localization of pDNA and transfection. Understanding in decomplexation and intracellular localization of pDNA may help develop more effective polyplexes.
Biomacromolecules 12/2012; · 5.48 Impact Factor
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ABSTRACT: No chemotherapeutic drug can be effective until it is delivered to its target site. Nano-sized drug carriers are designed to transport therapeutic or diagnostic materials from the point of administration to the drug's site of action. This task requires the nanoparticle carrying the drug to complete a journey from the injection site to the site of action. The journey begins with the injection of the drug carrier into the bloodstream and continues through stages of circulation, extravasation, accumulation, distribution, endocytosis, endosomal escape, intracellular localization and-finally-action. Effective nanoparticle design should consider all of these stages to maximize drug delivery to the entire tumor and effectiveness of the treatment.
Nano Today 12/2012; 7(6):606-618. · 15.35 Impact Factor
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ABSTRACT: For long-term, sustained protein delivery, a new, star-shaped block copolymer composed of methoxy poly(ethylene glycol) (mPEG), branched oligoethylenimine (bOEI), and poly(l-histidine) (pHis) was synthesized via the multi-initiation and ring-opening polymerization (ROP) of His N-carboxy anhydride (NCA) on bOEI with a PEG conjugation. The resulting mPEG-bOEI-pHis (POH) had strong buffering capacity within the neutral-to-acidic pH range and was complexed with insulin (Ins) via an electrostatic attraction plus hydrophobic interactions, resulting in the formation of a dual-interaction complex (DIC, weight ratio 2) of approximately 30-60 nm in size. This DIC tolerated high salt concentrations without destabilization, supporting the existence of hydrophobic interactions, and protected Ins from the organic solvent/water interface. The DIC in poly(lactide-co-glycolide) microspheres (PLGA MS) as a long-term Ins delivery formulation was evenly distributed via a double-emulsion method. The DIC-loaded PLGA MS offered a higher Ins loading and a lower initial burst than Ins-loaded PLGA MS. This formulation possessed near zero-order release kinetics (for at least one month). In streptozotocin (STZ)-induced diabetic rats, a DIC-loaded PLGA MS formulation was able to maintain blood-glucose levels at 200-350 mg/dL for the first two weeks and even lower levels (100-200 mg/dL) for the next two weeks. Thus, a new POH polymer and its complex with a drug protein could have potential biological application as a long-term, sustained protein delivery system.
Biomaterials 09/2012; 33(34):8848-57. · 7.40 Impact Factor
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ABSTRACT: To overcome the limitations of monomeric pH probes for acidic tumor environments, this study designed a mixed micelle pH probe composed of polyethylene glycol (PEG)-b-poly(l-histidine) (PHis) and PEG-b-poly(l-lactic acid) (PLLA), which is well-known as an effective antitumor drug carrier. Unlike monomeric histidine and PHis derivatives, the mixed micelles can be structurally destabilized by changes in pH, leading to a better pH sensing system in nuclear magnetic resonance (NMR) techniques. The acidic pH-induced transformation of the mixed micelles allowed pH detection and pH mapping of 0.2-0.3 pH unit differences by pH-induced "on/off"-like sensing of NMR and magnetic resonance spectroscopy. The micellar pH probes sensed pH differences in nonbiological phosphate buffer and biological buffers such as cell culture medium and rat whole blood. In addition, the pH-sensing ability of the mixed micelles was not compromised by loaded doxorubicin. In conclusion, PHis-based micelles could have potential as a tool to simultaneously treat and map the pH of solid tumors in vivo.
Biomacromolecules 08/2012; 13(9):2945-51. · 5.48 Impact Factor
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ABSTRACT: To deliver nucleic acids including plasmid DNA (pDNA) and short interfering RNA (siRNA), polymeric gene carriers equipped with various functionalities have been extensively investigated. The functionalities of these polymeric vectors have been designed to overcome various extracellular and intracellular hurdles that nucleic acids and their carriers encounter during their journey from injection site to intracellular target site. This review briefly introduces known extracellular and intracellular issues of nucleic acid delivery and their solution strategies. We examine significant yet overlooked factors affecting nucleic acid delivery (e.g., microenvironmental pH, polymer/siRNA complexation, and pharmaceutical formulation) and highlight our reported approaches to solve these problems.
Journal of Controlled Release 07/2012; · 5.73 Impact Factor
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ABSTRACT: The objective of this study was to investigate the efficiency of multifunctional poly(ethylene glycol)-based hemoglobin conjugates crosslinked with antioxidant enzymes for their ability to protect an oxygen carrier (hemoglobin) and insulin secreting islets from the combination of hypoxic and free radical stress under simulated transplantation conditions. In this study, RINm5F cells and isolated pancreatic islets were challenged with oxidants (H(2)O(2) or xanthine and xanthine oxidase) and incubated with conjugates (hemoglobin-hemoglobin or superoxide dismutase-catalase-hemoglobin) in normoxia (21% oxygen) or hypoxia (6% or 1% oxygen). Hemoglobin protection, intracellular free radical activity and cell viability in RINm5F cells measured by methemoglobin, dichlorofluorescein-diacetate, and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, respectively, showed that cells were better protected by conjugates containing antioxidant enzymes. Insulin secretion from islets and qualitative confocal evaluation of viability showed beta cells were protected by conjugates containing antioxidant enzymes when exposed to induced stress. Our study suggested that antioxidant enzymes play a significant role in hemoglobin protection and thus extended cell protection.
Biotechnology and Bioengineering 03/2012; 109(9):2392-401. · 3.95 Impact Factor
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ABSTRACT: Poly(lactide-co-glycolide) (PLGA) has most often been employed for the controlled release of protein formulations because of its safety profile with non-toxic degradation products. Nevertheless, such formulations have been plagued by a local acidic microenvironment and protein-polymer interactions, which result in chemical and physical denaturation of loaded proteins and often unfavorable release profiles. This study investigated the pH change of inner PLGA microsphere (MS) using charged (PLGA)(n)-b-branched polyethyleneimine (bPEI) micelles. The designed micelles can be transformed into either micelle or reverse micelle (RM) depending on the solvent and RM can form microspheres. In addition, (PLGA)(n)-b-bPEI can be modified into (PLGA)(n)-b-(carboxylated bPEI) via carboxylation of the primary amines. Cationic micelle (CM) or anionic micelle (AM) was complexed with counter-charged proteins leading to nanosized particles (approximately 100nm). In the micelle/protein complexes, the micelles mostly maintained their proton buffering capacity, and consequently, prevented or delayed the typical decrease in pH caused by degradation of PLGA in aqueous solution. Reconstitutable micelle/protein complexes allowed for increased and fine-tuned protein loading (~20wt.% when using CM1 (CM prepared from PLGA(36kDa)-b-bPEI(25kDa))/insulin complexes) in PLGA MS. In CM2 (CM prepared from (PLGA(36kDa))(2)-b-bPEI(25kDa))/insulin (4 of weight ratio (WR) of micelle to protein; WR4)-loaded PLGA MS, CM2 strongly prevented the micellar nanoenvironmental pH (pH 6.6 within 5days and then approximately pH 8.5) to be acidified in PLGA MS for 9weeks, unlike CM2-free PLGA MS. In conclusion, our findings propose that the proton buffering capacity and protein loading in PLGA MS can be tuned by controlling the complexation ratios of micelles and proteins, polymeric architectures of (PLGA)(n)-b-bPEI copolymers and WR of micelle/protein complexes and PLGA (or RM).
Journal of Controlled Release 03/2012; 160(3):440-50. · 5.73 Impact Factor
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ABSTRACT: Although polymers, polyplexes, and cells are exposed to various extracellular and intracellular pH environments during polyplex preparation and polymeric transfection, the impact of environmental pH on polymeric transfection has not yet been investigated. This study aims to understand the influence of environmental pH on polymeric transfection by modulating the pH of the transfection medium or the culture medium. Changes in the extracellular pH affected polymeric transfection by way of complex factors such as pH-induced changes in polymer characteristics (e.g., proton buffering capacity and ionization), polyplex characteristics (e.g., size, surface charge, and decomplexation), and cellular characteristics (e.g., cellular uptake, cell cycle phases, and intracellular pH environment). Notably, acidic medium delayed endocytosis, endosomal acidification, cytosolic release, and decomplexation of polyplexes, thereby negatively affecting gene expression. However, acidic medium inhibited mitosis and reduced dilution of gene expression, resulting in increased transfection efficiency. Compared to pH 7.4 medium, acidic transfection medium reduced gene expression 1.6-7.7-fold whereas acidic culture medium enhanced transfection efficiency 2.1-2.6-fold. Polymeric transfection was affected more by the culture medium than by the transfection medium. Understanding the effects of extracellular pH during polymeric transfection may stimulate new strategies for determining effective and safe polymeric gene carriers.
Biomaterials 11/2011; 33(5):1651-62. · 7.40 Impact Factor
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ABSTRACT: Tumors have been a highlight in the research of nanomedicine for decades. Despite all the efforts in the decoration of the nano systems, tumor specific targeting is still an issue due to the heterogeneous nature of tumors. Hypoxia is frequently observed in solid tumors. The consequent acidification of tumor extracellular matrices may bring new insight to tumor targeting. In this review, we present the polymeric nano systems that target tumor extracellular pH (pH(e)).
Colloids and surfaces. B, Biointerfaces 10/2011; 99:116-26. · 2.60 Impact Factor
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Journal of Controlled Release 09/2011; 155(2):113-4. · 5.73 Impact Factor
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ABSTRACT: The magnetic resonance (MR) functionalities of pullulan-conjugated gadolinium diethylene triamine pentaacetate (Gd-DTPA-Pullulan) as a new hepatocyte-specific contrast agent were evaluated. Pullulan, which specifically accumulates on hepatocytes via asialoglycoprotein receptors, was chemically linked with Gd-DTPA. Gd-DTPA-Pullulan displayed three times greater contrast enhancement than Gd-DTPA-BMA (Omniscan) in delayed MR imaging (MRI) on orthotopic rat hepatocarcinoma (HCC). This contrast effect lasted up to 24h. In particular, Gd-DTPA-Pullulan displayed a discriminative MR contrast on the regenerative and malignant hepatic nodules sequentially observed during the progress of cirrhotic HCC. Approximately 50% of injected Gd-DTPA-Pullulan was eliminated via the hepato-biliary system. IC(50) of Gd-DTPA-Pullulan on Chang liver cells was much higher than Gd-DTPA and Gd-DTPA-BMA (309.1 ± 11.2, 173.5 ± 15.5 and 49.4 ± 8.9 μM, respectively). Any significant toxicities of Gd-DTPA-Pullulan at the conventional dose on the rats weren't detected on histology studies. Gd-DTPA-Pullulan worked as a hepatocyte-specific MR contrast agent with increased MR functionalities and an acceptable safety profile setting the scene for future clinical trials.
Biomaterials 08/2011; 32(22):5187-94. · 7.40 Impact Factor
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ABSTRACT: Cationic polymers are potential intracellular carriers for small interfering RNA (siRNA). The short and rigid nature of an siRNA chain often results in larger and more loosely packed particles compared to plasmid DNA (pDNA) after complexing with carrier polycations, and in turn, poor silencing effects are seen against the target mRNAs. A helper polyanion, pDNA, was incorporated along with siRNA to form compact nanosized polyplexes. At C/A (cation/anion) ratios of 2 and 5, poly(l-lysine) (PLL)/siRNA-pGFP and PLL/siRNA-pGFP-OSDZ (oligomeric sulfadiazine (OSDZ) for endosomolysis) complexes produced particles 90-150 nm in size with a 15-45 mV surface charge, while PLL/siRNA complexes yielded particles 1-2 μm in size at the same C/A ratios. The PLL/siRNA-pGFP (C/A 2) complexes showed significantly higher specific gene silencing (50-90% vs. 10-25%) than the complexes formed at C/A 5. PLL/siRNA-pGFP-OSDZ (C/A 2) complexes improved the specific gene silencing (90%) more dramatically than PLL/siRNA-pGFP (C/A 2) complexes (50%), demonstrating a potential role for OSDZ. PLL/siRNA-pGFP-OSDZ (C/A 2) complexes sustained higher specific gene silencing compared with PLL/siRNA-pGFP (C/A 2) complexes. Other oligomeric sulfonamides (OSA) with varying pK(a) used in PLL/siRNA-pGFP-OSA complexes also caused effective gene silencing. The pGFP in the PLL/siRNA-pGFP complexes successfully expressed GFP protein without interfering with the siRNA. In conclusion, this study demonstrates that long pDNA helps effectively form nanosized siRNA particles and that OSA enhances specific gene silencing. In a single nucleic acid carrier formulation, co-delivery of siRNA and pDNA is feasible to maximize therapeutic effects or to include therapeutic or diagnostic functionalities.
Biomaterials 07/2011; 32(21):4914-24. · 7.40 Impact Factor
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Journal of Controlled Release 06/2011; 153(3):198-205. · 5.73 Impact Factor
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ABSTRACT: A low p50 hemoglobin (Hb) (p50 indicates O(2) tension at which Hb is half-saturated)-based oxygen carrier conjugated to antioxidant enzymes via dicarboxymethylated poly(ethylene glycol) (PEG) linker may have the beneficial effect in protecting pancreatic beta cells from severe hypoxia at transplantation sites. In this study, the oxygen dissociation curves, Hill plots, Bohr Effect, and oxygen content of Hb conjugates were measured. The protective effect due to incubation of Hb-conjugates (Hb/PEG molar ratio 1:10) with pancreatic beta cells (RINm5F) against hypoxia (6%, 3%, and 1% oxygen) was evaluated by an MTT assay and confocal microscopy. Quantitatively, Hb conjugates with antioxidant enzymes offered statistically significant protection (p<0.01, increased viability ∼80%) from hypoxia compared to control cells in 1% oxygen environment. Confocal images also showed that the low p50 Hb conjugates with antioxidants protected RINm5F cells from hypoxia.
Tissue Engineering Part A 05/2011; 17(19-20):2453-62. · 4.64 Impact Factor
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ABSTRACT: To study the development of porous poly(lactic-co-glycolic acid) microparticles (PLGA MPs) coated initially with albumin and then with palmityl-acylated exendin-4 (Pal-Ex4) as an inhalation system for treating diabetes.
Porous PLGA MPs were prepared by w/o/w double emulsification using hydroxypropyl-β-cyclodextrin and poly(ethylene-alt-maleic anhydride). Albumin was covalently attached to the MPs using EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide); Pal-Ex4 was then bound on the albumin surface. Albumin-binding degree and aerosolization efficiency were investigated. Deposition of the MPs after insufflations into the lungs of ICR mice was observed by image monitoring, and pulmonary hypoglycemic efficacies were examined in db/db mice. Cytotoxicity and histopathology induced by MPs were examined in Calu-3 and A549 cells and in the lungs of db/db mice, respectively.
Albumin-coating and Pal-Ex4-binding to porous MP were performed with acceptable efficiencies. Pal-Ex4-bound albumin-coated MPs (Pal-Ex4/HSA-PLGA MP) were of high porosity and had appropriate aerodynamic sizes. Furthermore, this MP was efficiently deposited throughout mouse lungs, and exhibited a prolonged hypoglycemia and no significant lung tissue damage in db/db mice.
Pal-Ex4/HSA-PLGA MP demonstrated many meaningful pharmaceutical advantages for the treatment of diabetes, in terms of aerosolization efficiency, drug loading, sustained drug-release, and hypoglycemic duration in vivo.
Pharmaceutical Research 04/2011; 28(8):2008-19. · 4.09 Impact Factor
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ABSTRACT: The controlled release of drugs from hydrogels has progressed from systems that merely swell in water, to novel hydrogels with specific chemical properties which respond to various stimuli to facilitate drug release.Specifically, electro-erodible hydrogels are synthesized by polymer chain complexation and precipitation through hydrogen or ionic bonds. The surface of the solid polymer device is eroded or dissolved in response to an applied electric current. When a constant electric current is applied, zero-order release of entrapped drug is achieved. However, if a step-function current is applied, the drug can be released in an on-off pattern.Concanavalin A microspheres, a protein gel, has been synthesized to release insulin in response to glucose concentrations. This microsphere gel has specific binding affinity for glucose and mannose. A synthesized glycosylated insulin was loaded on the microspheres and then released in response to glucose concentrations via a competitive binding exchange mechanism.
Macromolecular Symposia 03/2011; 70-71(1):173 - 181.
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ABSTRACT: As an approach to replacing islets of Langerhans in an implanted biohybrid artificial pancreas, thermally reversible polymers based on N-isopropylacrylamide were synthesized and then evaluated as an extracellular matrix for islets in an immunoprotecting membrane pouch. A high molecular weight poly(N-isopropylacrylamide-co-acrylic acid (2 mole % in feed)) demonstrated gelation at 37°C and became a solution below 30°C. This polymer exhibited minimum syneresis (water separation) upon gelation from a solution state when the temperature was raised from room temperature to 37°C, while poly(N-isopropylacrylamide) exhibited considerable syneresis under the same conditions. These properties influence the efficiency of islet entrapment. The copolymer was able to entrap rat islets almost 100%, but the homopolymer entrapped less than 50%. The static insulin secretion of the islets in the copolymer matrix at high glucose concentration (16.5 mM) was comparable to that of control islets, however, the entrapped islets showed prolonged viability in vitro. These results indicate the potential of developing a rechargeable biohybrid pancreas using thermally reversible polymer gels.
Macromolecular Symposia 03/2011; 109(1):155 - 167.
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ABSTRACT: This study investigated the potential of creating a charged polymeric micelle-based nucleic acid delivery system that could easily be reconstituted by the addition of water. (PLGA(36kDa))(2)-b-bPEI(25kDa) (PLGA MW 36 kDa, bPEI M(w) 25 kDa, PLGA:bPEI block ratio = 2) was synthesized and used to prepare cationic micelles. The copolymer retained proton-buffering capability from the bPEI block within the endosomal pH range. Micelle/pDNA complexes retained their particle size (100-150 nm) and surface charge (30-40 mV) following reconstitution. It was found that adding a small amount of low molecular weight bPEI (1.8 kDa) completely shielded pDNA in the micelle/pDNA complexes and enhanced transfection efficiency 50-100 fold for both fresh and reconstituted complexes without affecting complex size. Transfection efficiency for "reconstituted" micelle/pDNA/bPEI(1.8kDa) (WR 1) complexes was 16-fold higher than its "fresh" counterpart. Although transfection levels achieved using "reconstituted" micelle/pDNA/bPEI(1.8kDa) complexes were 3.6-fold lower than control "fresh" bPEI(25kDa)/pDNA (N/P 5) complexes, transfection levels were 39-fold higher than "reconstituted" bPEI(25kDa)/pDNA (N/P 5) complexes. The micelle/pDNA/bPEI(1.8kDa) system showed very low cytotoxicity in MCF7 cells even with pDNA doses up to 20 μg, and transfection levels increased linearly with increasing pDNA dose. These results indicate that this PLGA-b-bPEI polymeric micelle-based system is well suited as a reconstitutable gene delivery system, and has high potential for use as a delivery system for gene therapy applications.
Biomaterials 02/2011; 32(15):3845-54. · 7.40 Impact Factor
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ABSTRACT: This study primarily focused on the anti-metastatic activity of doxorubicin (DOX) loaded in a pH-sensitive mixed polymeric micelle formed from two block polymers: poly(L-lactide) (PLLA) (Mn 3000)-b-poly(ethylene glycol) (PEG) (Mn 2000)-folate and poly(L-histidine) (PHis) (Mn 4700)-b-PEG (Mn 2000). Tumor formation and metastasis in mice were examined using a murine mammary carcinoma cell of 4T1 which is one of the most aggressive metastatic cancer cell lines. The efficacy was evaluated by tumor size, body weight change, survival rate, dorsal skin fold window chamber model, and histological observation of the lung, heart, liver and spleen after treatment with various DOX formulations. When the tumor reached 50-100 mm³ in size, the mice were treated 4 times at a 3-day interval at a dose of 10 mg DOX/kg. The mixed micelle formulation resulted in retarded tumor growth, no weight loss, and no death for 4-5 weeks. In another set of the in vivo test for histological evaluation of the organs, the mice were similarly treated but the formulations were injected one day after 4T1 cell inoculation. The treatment by DOX loaded mixed micelle showed no apparent metastasis till 28 days. However, significant metastasis to the lung and heart was observed on Day 28 when the mice were treated with DOX carried by PBS, PLLA-b-PEG micelle and PHis-b-PEG micelle.
Journal of Controlled Release 02/2011; 152(1):84-9. · 5.73 Impact Factor
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ABSTRACT: Hemoglobin (Hb) conjugated with the antioxidant enzymes (SOD and CAT), by employing dicarboxymethylated poly(ethylene glycol), was designed for protection of hemoglobin against free radicals. In this study, the conjugation process was confirmed by employing SDS-PAGE and SEC techniques. The average molecular weight of the conjugates was estimated to be around 1000 kDa. The enzymatic activities of the SOD and CAT in the conjugates (Hb-SOD-CAT) after conjugation were found to retain greater than 70% and 90% of the original bioactivity. Results show that antioxidant enzymes helped minimize methemoglobin (non-carrier of oxygen) formation during the conjugation process and also during storage at 4°C over a period of 1 month. In summary, the optimized (1:10 Hb/PEG) crosslinked conjugates with antioxidant enzymes showed protective properties from severe free radical stresses when incubated with hydrogen peroxide (0.1 and 1 mM) and xanthine (1 mM)/xanthine oxidase (10 and 20 mU/ml) system.
International journal of biological macromolecules 12/2010; 47(5):603-13. · 2.37 Impact Factor
