Shengxiang Fu

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. STATEMENT OF SIGNIFICANCE Chemotherapeutic 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.

Acid–degradable carboxymethyl chitosan nanogels via an ortho ester linkage mediated improved penetration and growth inhibition of 3-D tumor spheroids in vitro

This work describes an acid-degradable and tumor-targeted nanogels prepared by the copolymerization between lactobionic acid-modified methacrylated carboxymethyl chitosan and acid-labile methacrylated orthoester-based monomers. The size distribution and micromorphology of the prepared nanogels were observed by dynamic light scattering, transmission electron microscopy and scanning electron microscopy. The stability of nanogels in various environments was then investigated. Doxorubicin as a model drug was successfully encapsulated into nanogels. In vitro cellular uptake and MTT results indicate that the tumor-targeting and pH-sensitive nanogels display higher cellular internalization and cytotoxicity than non-target nanogels and free DOX. The improved penetration and growth inhibition against 3-D multicellular spheroids further demonstrate that the dual-functional nanogels may be a potential nano-carrier for drug delivery in cancer therapy.

Hyaluronic acid nanogels prepared via ortho ester linkages show pH-triggered behavior, enhanced penetration and antitumor efficacy in 3-D tumor spheroids

A new type of pH-triggered hyaluronic acid nanogel system (HA-NGs) was successfully developed for tumor-targeted delivery of drugs. HA-NGs were obtained by copolymerization between methacrylate HA and a new cross-linker containing ortho ester groups in an aqueous solution. The therapeutic drug (DOX) was loaded into the HA-NGs (DOX@HA-NGs) and exhibited appropriate loading of about 17.3% with a size of around 200nm. Such new pH-triggered HA-NGs are found to be highly desirable for targeted cancer therapy because it could significantly minimize the amount of premature drug release in neutral pH, and also provide a sufficient amount of drug to effectively kill the cancer cells caused by the degradation of ortho ester groups at acid pH values. Results from the cellular uptake and cytotoxicity of DOX@HA-NGs performed in two-dimensional (2D) cell culture demonstrated that DOX@HA-NGs exhibit excellent tumor homing and higher cytotoxicity. Importantly, the penetration and inhibition against three-dimensional (3D) tumor spheroids demonstrated that DOX@HA-NGs could fully penetrate into HepG2 tumor spheroids, thus leading to higher inhibition. So, such new tumor-targeting DOX@HA-NGs prepared via ortho ester linkages will exhibit excellent stability in a neutral environment, pH-triggered drug release, as well as enhanced penetration and destruction against 3D tumor spheroids, thereby making targeted cancer therapy possible.

pH-triggered chitosan nanogels via an ortho ester-based linkage for efficient chemotherapy

We report on new types of chitosan-based nanogels via an ortho ester-based linkage, used as drug carriers for efficient chemotherapy. First, we synthesized a novel diacrylamide containing ortho ester (OEAM) as an acid-labile cross-linker. Subsequently, methacrylated succinyl-chitosan (MASCS) was prepared and polymerized with OEAM at different molar ratios to give a series of pH-triggered MASCS nanogels. Doxorubicin (DOX) as a model anticancer drug was loaded into MASCS nanogels with a loading content of 16.5%. As expected, with the incorporation of ortho ester linkages, these nanogels showed pH-triggered degradation and drug release at acidic pH values. In vitro cellular uptake shows that the DOX-loaded nanogels could be preferentially internalized by two-dimensional (2D) cells and three-dimensional (3D) multicellular spheroids (MCs), resulting in higher inhibition of the proliferation of tumor cells. In vivo biodistribution and anti-tumor effect were determined in H22 tumor-bearing mice, and the results demonstrate that the acid-labile MASCS nanogels can significantly prolong the blood circulation time of DOX and improve the accumulation in tumor areas, leading to higher therapeutic efficacy. STATEMENT OF SIGNIFICANCE We designed new pH-triggered chitosan nanogels via an ortho ester-based cross-linker for efficient drug-loading and chemotherapy. These drug-loaded nanogels exhibit excellent pH-triggered drug release behavior due to the degradation of ortho ester linkages in mildly acidic environments. In vitro and in vivo results demonstrate that the nanogels could be efficiently internalized by 2D cells and 3D-MCs, improve drug concentration in solid tumors, and lead to higher therapeutic efficacy. To the best of our knowledge, this is the first report on using an ortho ester-based cross-linker to prepare pH-triggered chitosan nanogels as tumor carriers, which may provide a potential route for improved safety and to increase the therapeutic efficacy of anticancer therapy.

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.

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.

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 100–200 nm, and exhibited much lower cytotoxicity against 293T and SH-SY5Y cells than that of branched polyethylenimine (25 kDa). 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 (25 kDa). Furthermore, these SP-PEIs/DNA polyplexes could effectively transfect 293T and SH-SY5Y cells with or without 10% serum, suggesting their excellent serum tolerance.

Low molecular weight PEI-grafted carboxyl-modified soybean protein as gene carriers with reduced cytotoxicity and greatly improved transfection in vitro

Abstract SA-SP-PEI were synthesized via acylation reaction between carboxyl-modified soybean protein (SA-SP) and branched polyethylenimine (PEI) with molecular weight of 600, 1200, and 1800 Da, and designed as SA-SP-PEI600, SA-SP-PEI1200, and SA-SP-PEI1800, respectively. SA-SP-PEI could effectively condense plasmid DNA into nanoscale polyplexes and protect them from enzymatic digestion. MTT assay revealed that SA-SP-PEI exhibited reduced cytotoxicity on 293 T and SH-SY5Y cells. SA-SP-PEI1800/DNA complexes hold highest transfection efficiency on 293 T and SH-SY5Y cells with or without 10% serum, which was owing to its better serum stable and improved biocompatibility. Such polycationic soybean proteins have great potentials as gene carriers by further optimization. Graphical Abstract

pH-triggered poly(ethylene glycol) nanogels prepared through orthoester linkages as potential drug carriers

ABSTRACT The new acid-labile poly(ethylene glycol) (PEG) nanogels were prepared by copolymerization of a new crosslinking agent containing orthoester groups (OEAM) and methoxypolyethylene glycol acrylate (MPEGAC). DOX was loaded into PEG nanogels with a loading content of 18.2%, which was highly desirable for targeted cancer therapy without premature drug release in neutral environment. The cellular uptake and cytotoxicity of DOX-loaded PEG nanogels were measured using SH-SY5Y and HepG2 cells. Tumor penetration and antitumor activity were investigated using SH-SY5Y tumor-like spheroids. All results demonstrate that the pH-sensitive PEG nanogels may be used as potential drug carriers for chemotherapy. GRAPHICAL ABSTRACT

Acid-degradable lactobionic acid-modified soy protein nanogels crosslinked by ortho ester linkage for efficient antitumor in vivo

Graphical abstract Figure. No caption available. Abstract It remains a crucial challenge to achieve efficient cellular uptake and intracellular drug release in tumor cells for the nanoscale drug delivery systems. Herein, acid‐degradable nanogels were prepared by cross‐linking methacrylated soy protein with an acid‐labile ortho ester cross‐linker (NG1), and then modified with lactobionic acid (LA) to give tumor‐targeted nanogels (NG2). Both NG1 and NG2 displayed excellent stability in neutral environment, while showed pH‐triggered degradation behaviors under mildly acidic conditions resulting from the breakage of ortho ester bonds. Doxorubicin (DOX) was successfully loaded into nanogels, which exhibited an accelerated release at low pH. In vitro cell studies demonstrated that LA‐modified nanogels could effectively improve cellular internalization, show higher cytotoxicity and apoptosis toward asialoglycoprotein receptor (ASGPR) over‐expressed HepG2 cells. In vivo antitumor experiment proved that LA modification could significantly enhance the tumor‐targeting ability of nanogels and increase DOX concentration in tumor site, leading to better therapeutic efficacy. Histological analysis further demonstrated that soy protein‐based nanogels did not cause any damage to normal organs. Overall, these pH‐sensitive and tumor‐targeting soy protein‐based nanogels can be potential drug carriers for efficient tumor treatment.