Guoqing Yan

Low Molecular Weight PEI-Based Vectors via Acid-Labile Ortho Ester Linkage for Improved Gene Delivery

A series of novel pH-sensitive gene delivery vectors (POEI 1, 2, and 3) are synthesized through Michael addition from low molecular weight PEI (LMW PEI) via acid-labile ortho ester linkage with terminal acrylates (OEAc) by various feed molar ratios. The obtained POEI 1 and POEI 2 can efficiently condense plasmid DNA into nanoparticles with size range of 200-300 nm and zeta-potentials of about +15 mV while protecting DNA from enzymatic digestion compared with POEI 3. Significantly, ortho ester groups of POEI main-chains can make an instantaneous degradation-response to acidic endosomal pH (≈5.0), resulting in accelerated disruption of polyplexes and intracellular DNA release. MTT assay reveals that all POEIs exhibit much lower cytotoxicity in different cells than branched PEI (25 KDa). As expected, POEI 1 and POEI 2 perform improved gene transfection in vitro, suggesting that such polycations might be promising gene vectors based on overcoming toxicity-efficiency contradiction.

Stepwise targeted drug delivery to liver cancer cells for enhanced therapeutic efficacy by galactose-grafted, ultra-pH-sensitive micelles.

To promote drug accumulation and cell-killing ability at tumor tissue, we have prepared a stepwise targeted drug delivery system that can remain stealthy and long-circulating in the blood vessels, improve drug retention at extracellular stimuli, enhance cellular uptake through special targeting ligands, and then achieve rapid drug release to improve toxicity to tumor cells at intracellular stimuli. Herein, galactose-grafted, ultra-pH-sensitive drug carriers (POEAd-g-LA-DOX micelles), which could respond to both extracellular and intracellular pH, and combine with galactose-receptors in cell membrane, were constructed by a facile method, therefore achieving: (i) remaining stable at pH 7.4; (ii) responding to tumoral extracellular pH following gradually larger nanoparticles (NPs); (iii) conjugating receptors in the cell membrane of liver cancer through surface galactose-ligands of micelles; (iv) being sensitive to tumoral intracellular pH following further swelling for rapid drug release. In vitro cytotoxicity and cellular uptake measurement showed that POEAd-g-LA20-DOX micelle was more easily internalized and more toxic effect on tumor cells than free DOX. Moreover, in vivo biodistribution and tumor inhibition examinations demonstrated that POEAd-g-LA20-DOX formulation had more superior efficacy to significantly enhance drug accumulation in tumor, and then restrain tumor growth while decreasing drug concentration in heart.nnnSTATEMENT OF SIGNIFICANCEnChemotherapeutic efficacy is limited by poor tumor selectivity, which also causes severe toxicity in normal tissues and organs, although many targeted drug delivery systems have been developed by passive targeting strategies or active targeting strategies with specific targeting ligands in recent years. Herein, galactose-grafted, ultra-pH-sensitive, ortho ester-based drug carriers, which can respond to both extracellular and intracellular pH, and target to galactose-receptors in cell membrane, have been successfully constructed by facile method, therefore achieving stepwise targeting to microenvironment of liver cancer and then enhancing drug accumulation and tumor inhibition. The strategy of designing dual-stimuli-responsive copolymers can be potentially useful, and extrapolated to synthesizing other categories of highly labile drug carriers in a range of biomedical applications.

Tunable dynamic fluorinated poly(orthoester)-based drug carriers for greatly enhanced chemotherapeutic efficacy

To promote chemotherapeutic efficacy, a dynamic drug delivery system was prepared, which remained stealthy and stable in blood vessels, improved drug penetration and retention in response to extracellular stimuli, enhanced membrane permeability through unique functional groups, and then achieved rapid drug release to improve toxicity to tumor cells in response to intracellular stimuli. Herein, fluorinated poly(orthoester)-based nanospheres-doxorubicin (POEAd-g-F-DOX), which were constructed by a facile method, can respond to both extracellular and intracellular pH in a controlled manner and can easily pass through cell membranes by fluorination, are reported for the first time as drug carriers; they demonstrate (i) stability at pH 7.4; (ii) response to tumoral extracellular pH following dynamic size changes; (iii) crossing of cell membranes; and (iv) sensitivity to tumoral intracellular pH following dissolution or further swelling for rapid drug release. POEAd-g-F-DOX exhibits greatly enhanced chemotherapeutic efficacy because of its tunable dynamic changes and fluorination, resulting in improved cellular uptake and greater toxic effects on tumor cells than free DOX, stronger penetration and unprecedentedly rapid complete destruction of 3-D tumor spheroids in vitro, and significant tumor accumulation and inhibition with decreasing side effects in normal organs in vivo.

Well-Defined Poly(Ortho Ester Amides) for Potential Drug Carriers: Probing the Effect of Extra- and Intracellular Drug Release on Chemotherapeutic Efficacy

To compare the chemotherapeutic efficacy determined by extra- and intracellular drug release strategies, poly(ortho ester amide)-based drug carriers (POEAd-C) with well-defined main-chain lengths, are successfully constructed by a facile method. POEAd-C3-doxorubicin (DOX) can be rapidly dissolved to release drug at tumoral extracellular pH (6.5-7.2), while POEAd-C6-DOX can rapidly release drug following gradual swelling at intracellular pH (5.0-6.0). In vitro cytotoxicity shows that POEAd-C3-DOX exhibits more toxic effect on tumor cells than POEAd-C6-DOX at extracellular pH, but POEAd-C6-DOX has stronger tumor penetration and inhibition in vitro and in vivo tumor models. So, POEAd-C6-DOX with the intracellular drug release strategy has stronger overall chemotherapeutic efficacy than POEAd-C3-DOX with extracellular drug release strategy. It is envisioned that these poly(ortho ester amides) can have great potential as drug carriers for efficient chemotherapy with further optimization.

pH-sensitive nanogels with ortho ester linkages prepared via thiol-ene click chemistry for efficient intracellular drug release

pH-sensitive nanogels with ortho ester linkages were conveniently prepared through reaction of thiol-ene click chemistry. Through adjusting feed reactant ratios and concentrations of ortho ester diacrylamide (OEAM), pentaerythritol tetra(3-mercaptopropionate) (PT), and methoxyl poly(ethyleneglycol) acrylate (mPEGA), the size of the nanogels could be controlled at 100-200nm with relatively narrow size distributions. The nanogels with size of 149.1±17.7nm (designed as NG) were verified by proton nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FT-IR), dynamic laser scattering (DLS) and transmission electron microscopy (TEM). Doxorubicin (DOX) was loaded into NG with high drug loading efficiency up to 73.7%. In vitro drug release studies showed that up to 75.9% DOX from NG was released in 24h at pH 5.0 because of hydrolysis of ortho ester. Cellular uptake studies confirmed that DOX-loaded NG (NG/DOX) could be readily internalized by two-dimensional cells, resulting in efficient antitumor efficiency of cancer cells. Three-dimensional (3D) multicellular tumor spheroids (MCTS) as in vitro tumor model was used to further evaluate the antitumor effect of NG/DOX. The results demonstrated that NG/DOX showed a significantly enhanced penetration and growth inhibition in 3D multicellular tumor spheroids (MCTS), compared to free DOX.

Dual-stimuli-sensitive poly(ortho ester disulfide urethanes)-based nanospheres with rapid intracellular drug release for enhanced chemotherapy

Herein, new poly(ortho ester disulfide urethanes) (POEDU) and poly(ortho ester urethanes) (POEU) were successfully synthesized via polycondensation between active esters of 1,6-hexandiol (HD) and dual-stimuli-sensitive ortho ester disulfide diamine or pH-senstive ortho ester diamine. The corresponding POEDU and POEU nanospheres were easily fabricated using an oil-in-water emulsion technique. In vitro degradation experiments indicated that POEDU nanospheres degraded faster than POEU nanospheres in mildly acidic and reductive environments. Doxorubicin (DOX) as a model antitumor drug was successfully incorporated into these nanospheres to give DOX-loaded nanoparticles (POEDU-DOX and POEU-DOX). In vitro drug release studies showed that release of DOX from dual-stimuli-sensitive POEDU-DOX was accelerated compared with release from the pH-sensitive POEU-DOX under DL-dithiothreitol (DTT) and mildly acidic conditions. In addition, in vitro uptake and cytotoxicity assays revealed that POEDU-DOX exhibited more efficient antitumor effect than POEU-DOX did against both two-dimensional (2D) cells and three-dimensional (3D) multicellular tumor spheroids (MCTS). Finally, in a mice H22 tumor model, POEDU-DOX exhibited preferable antitumor capability. In conclusion, the pH and redox dual-stimuli-sensitive POEDU nanospheres can be superior drug carriers for cancer treatment.

Hybrid pH-sensitive nanogels surface-functionalized with collagenase for enhanced tumor penetration

Densely extracellular matrix (ECM) is one of the main barriers that hinder the penetration of drug into tumor parenchyma, compromising the therapeutic activity. In this work, alginic acid was copolymerized with an acid-labile monomer to give the acid-degradable nanogels (ALG NG), which was then immobilized with collagenase to obtain the surface-functionalized nanogels (Co@ALG NG). The introduction of collagenase would enhance the diffusion ability of nanogels in tumor parenchyma based on the hydrolytic activity to tumor ECM. The stability of these nanogels in various physiological environment and the pH-triggered degradation and drug release at different pH were then investigated. Monolayer cell and tumor-like spheroids were used to illustrate the penetration and drug delivery. In vivo drug distribution and antitumor efficiency of these nanogels were investigated using H22 tumor-bearing mice. The immobilization of collagenase significantly enhance the nanogels penetration and diffusion ability in tumor area upon the digestion of tumor ECM, leading to higher drug concentration and superior antitumor effect.

Low molecular weight polyethylenimine-grafted soybean protein gene carriers with low cytotoxicity and greatly improved transfection in vitro:

A series of gene carriers (SP-PEI) have been synthesized by acylation reaction between soybean protein and branched polyethylenimine with low molecular weight of 600, 1200 and 1800 Da, and designed as SP-PEI600, SP-PEI1200 and SP-PEI1800, respectively. SP-PEI could effectively condense plasmid DNA into nanoscale polyplexes with size range of 100–200u2009nm, and exhibited much lower cytotoxicity against 293T and SH-SY5Y cells than that of branched polyethylenimine (25u2009kDa). In vitro gene transfection demonstrated that SP-PEI/DNA complex displayed increased transfection against 293T and SH-SY5Y cells with the increase of the weight ratio of SP-PEI/DNA complex with or without 10% serum. At weight ratio of 24, SP-PEI1800/DNA polyplexes showed the highest transfection on SH-SY5Y cells, which was almost three folds higher than PEI (25u2009kDa). Furthermore, these SP-PEIs/DNA polyplexes could effectively transfect 293T and SH-SY5Y cells with or without 10% serum, suggesting their excellent serum tolerance.

Bromelain-immobilized and lactobionic acid-modified chitosan nanoparticles for enhanced drug penetration in tumor tissues

Abstract Tumor extracellular matrix (ECM) still remains a major barrier that hinders the diffusion of drug in tumor parenchyma. We design a lactobionic acid (LA) and bromelain dual-functional nanoparticles as doxorubicin carriers. LA can increase the nanoparticles tumor-targeting ability, while bromelain can enhance the permeation of nanoparticles in tumor parenchyma due to the hydrolytic activity to ECM. The chemical structures of these modified macromolecules were characterized by 1 H NMR, FT-IR and UV spectrum. Drug-loaded nanoparticles as final drug formulations were observed in terms of size, zeta potential and micromorphology. Monolayer cells and three-dimensional cell spheroids were used to investigate the influence of LA and bromelain on the nanoparticles cellular uptake, cytotoxicity, accumulation and penetration. In vivo drug distribution and antitumor activity results demonstrated that bromelain increased nanoparticles penetration and diffusion ability in tumor area upon the digestion of ECM, leading to higher drug concentration in tumor area and superior antitumor effect.

TPGS-functionalized and ortho ester-crosslinked dextran nanogels for enhanced cytotoxicity on multidrug resistant tumor cells

Herein pH-sensitive nanogels (NG1) and P-glycoprotein-repressive nanogels (NG2) were prepared by copolymerization between an ortho ester crosslinker (OEAM) and tocopheryl polyethylene glycol succinate (TPGS)-free or conjugated dextran. Nanogels with or without TPGS possessed a uniform diameter (∼180u202fnm) and excellent stability in various physiological environments. Doxorubicin (DOX) was successfully loaded into NG1 and NG2 to give NG1/DOX and NG2/DOX, both of them showed appropriate drug release profiles under mildly acidic conditions (pH 5.0). NG2/DOX possessed higher drug enrichment and lethality than NG1/DOX did on MCF-7/ADR cells. Analysis of corresponding index of efflux activity showed that NG2 could induce depolarization of mitochondrial membrane and interfere with ATP metabolism. NG2/DOX also displayed increased penetration and growth inhibition on MCF-7/ADR multicellular spheroids. These results demonstrated that pH-sensitive TPGS-functionalized nanogels (NG2) as drug carriers had great potential to suppress drug efflux in MCF-7/ADR cells and even overcome MDR on cancer cells.