Microengineered 3D cell-laden thermoresponsive hydrogels for mimicking cell morphology and orientation in cartilage tissue engineering.

Mimicking the zonal organization of native articular cartilage, which is essential for proper tissue functions, has remained a challenge. In this study, a thermoresponsive copolymer of chitosan-g-poly(N-isopropylacrylamide) (CS-g-PNIPAAm) was synthesized as a carrier of mesenchymal stem cells (MSCs) to provide a support for their proliferation and differentiation. Microengineered three-dimensional (3D) cell-laden CS-g-PNIPAAm hydrogels with different microstripe widths were fabricated to control cellular alignment and elongation in order to mimic the superficial zone of natural cartilage. Biochemical assays showed six- and sevenfold increment in secretion of glycosaminoglycans (GAGs) and total collagen from MSCs encapsulated within the synthesized hydrogel after 28 days incubation in chondrogenic medium. Chondrogenic differentiation was also verified qualitatively by histological and immunohistochemical assessments. It was found that 75 ± 6% of cells encapsulated within 50 µm wide microstripes were aligned with an aspect ratio of 2.07 ± 0.16 at day 5, which was more organized than those observed in unpatterned constructs (12 ± 7% alignment and a shape index of 1.20 ± 0.07). The microengineered constructs mimicked the cell shape and organization in the superficial zone of cartilage whiles the unpatterned one resembled the middle zone. Our results suggest that microfabrication of 3D cell-laden thermosensitive hydrogels is a promising platform for creating biomimetic structures leading to more successful multi-zonal cartilage tissue engineering. Biotechnol. Bioeng. 2017;114: 217-231. © 2016 Wiley Periodicals, Inc.

Intracellular microenvironment responsive polymers: a multiple-stage transport platform for high-performance gene delivery.

A new strategy for promoting endoplasmic gene delivery and nucleus uptake is proposed by developing intracellular microenvironment responsive biocompatible polymers. This delivery system can efficiently load and self-assemble nucleic acids into nano-structured polyplexes at a neutral pH, release smaller imidazole-gene complexes from the polymer backbones at intracellular endosomal pH, transport nucleic acids into nucleus through intracellular environment responsive multiple-stage gene delivery, thus leading to a high cell transfection efficiency.

3D printing of cell-laden electroconductive bioinks for tissue engineering applications.

Bioprinting is an emerging powerful fabrication method, which enables the rapid assembly of 3D bioconstructs with dispensing cell-laden bioinks in pre-designed locations. However, to translate this technology into real applications, there are still a number of challenges that need to be addressed. First, the current inks are generally composed of polymeric materials with poor electrical conductivity that mismatches with the native tissue environment. The second challenge associated with the 3D bioprinting of hydrogel-based bioinks is the fabrication of anatomical-size constructs without any loss of shape fidelity and resolution. To address these challenges, in this work, we introduced a biocompatible bioink associated with current 3D bioprinting by combining methylcellulose and kappa-carrageenan (MC/κCA) hydrogels with poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) conducting polymers. The prepared ink exhibited highly thixotropic behaviour, which could be tuned via changing the concentration of MC and κCA to obtain easy printing with high shape fidelity. The ink was able to fabricate physiological-scale constructs without requiring a secondary support bath. In addition, varying the concentration of PEDOT:PSS could control the electrical conductivity of the ink. Moreover, the encapsulated human embryonic kidney 293 (HEK-293) cells in bulk hydrogels and 3D bioprinted structures maintained high cell viability (>96%) over a week, confirming the in vitro biocompatibility of the ink. Overall, these findings indicate that the MC/κCA/PEDOT:PSS bioink can be promising in biomedical applications, which improved the electroconductivity of bioinks and can exploit the advantage of conductive polymers in the 3D bioprinting technology.

Tuning microenvironment for multicellular spheroid formation in thermo-responsive anionic microgel scaffolds.

Tumor spheroids are considered to be effective in drug screening and evaluation. Three-dimensional scaffold-based cell culture becomes very promising in producing multicellular spheroids. Different from other approaches, 3D scaffolds mimic in vivo cellular microenvironment which encourages intercellular and extracellular interactions. The properties of the cellular microenvironment include the surface wettability, chemistry, and charge of the scaffolds which may influence cell attachment, proliferation as well as migration and these properties are essential for multicellular spheroids formation. Through co-polymerization with different carboxylic acids, we demonstrate that the surface charge density and hydrophobicity of the microenvironment have a great impact on the tumor spheroids formation progress and their size distribution. Our results show that a scaffold with a moderate negative charge density and a highly hydrophilic surface promotes cell proliferation, resulting in quicker and larger spheroids formation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2899-2909, 2018.

NIPAM-based Microgel Microenvironment Regulates the Therapeutic Function of Cardiac Stromal Cells.

To tune the chemical, physical, and mechanical microenvironment for cardiac stromal cells to treat acute myocardial infarction (MI), we prepared a series of thermally responsive microgels with different surface charges (positive, negative, and neutral) and different degrees of hydrophilicity, as well as functional groups (carboxyl, hydroxyl, amino, and methyl). These microgels were used as injectable hydrogels to create an optimized microenvironment for cardiac stromal cells (CSCs). Our results indicated that a hydrophilic and negatively charged microenvironment created from poly( N-isopropylacrylamide- co-itaconic acid) was favorable for maintaining high viability of CSCs, promoting CSC proliferation and facilitating the formation of CSC spheroids. A large number of growth factors, such as vascular endothelial growth factor (VEGF), insulin-like growth factor I (IGF-1), and stromal-derived factor-1 (SDF-1) were released from the spheroids, promoting neonatal rat cardiomyocyte activation and survival. After injecting the poly( N-isopropylacrylamide- co-itaconic acid) microgel into mice, we examined their acute inflammation and T-cell immune reactions. The microgel itself did not elicit obvious immune response. We then injected the same microgel-encapsulated with CSCs into MI mice. The result revealed the treatment-promoted MI heart repair through angiogenesis and inhibition of apoptosis with an improved cell retention rate. This study will open a door for tailoring poly( N-isopropylacrylamide)-based microgel as a delivery vehicle for CSC therapy.

Poly(N-isopropylacrylamide) hydrogel/chitosan scaffold hybrid for three-dimensional stem cell culture and cartilage tissue engineering.

Providing a controllable and definable three-dimensional (3D) microenvironment for chondrogenic differentiation of mesenchymal stem cells (MSCs) remains a great challenge for cartilage tissue engineering. In this work, poly(N-isopropylacrylamide) (PNIPAAm) polymers with the degrees of polymerization of 100 and 400 (NI100 and NI400) were prepared and the polymer solutions were introduced into the preprepared chitosan porous scaffolds (CS) to form hybrids (CSNI100 and CSNI400, respectively). SEM images indicated that the PNIPAAm gel partially occupied chitosan pores while the interconnected porous structure of chitosan was preserved. MSCs were incorporated within the hybrid and cell proliferation and chondrogenic differentiation were monitored. After 7-day incubation of the cell-laden constructs in a growth medium, the cell viability in CSNI100 and CSNI400 were 54 and 108% higher than that in CS alone, respectively. Glycosaminoglycan and total collagen contents increased 2.6- and 2.5-fold after 28-day culture of cell-laden CSNI400 in the chondrogenic medium. These results suggest that the hybrid structure composed of the chitosan porous scaffold and the well-defined PNIPAAm hydrogel, in particular CSNI400, is suitable for 3D stem cell culture and cartilage tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2764-2774, 2016.

Influence of polymer molecular weight on the in vitro cytotoxicity of poly (N-isopropylacrylamide).

Poly (N-isopropylacrylamide) (PNIPAAm) is a thermosensitive polymer with various biomedical applications. We examined molecular weight (MW)-dependent cytotoxicity of PNIPAAm. Our results indicated that low-MW PNIPAAm (degree of polymerization (DP)=35) is inherently toxic to cells. Moderate-MW PNIPAAms with their DP between 100 and 200 are non-cytotoxic. When cells are seeded on top of a polymer-coated surface, PNIPAAm with a higher MW (DP=400) shows non/low cytotoxicity, while when monolayer cells are exposed to the polymer solution, cell viability drops drastically. This may be due to lack of nutrient and oxygen rather than intrinsic toxicity of the polymer.

Application of response surface methodology to the modeling of alpha-amylase purification by aqueous two-phase systems.

Mathematical models concerning the purification of alpha-amylase from the cultivation supernatant of Bacillus subtilis in a polyethylene glycol-citrate aqueous two-phase system (ATP) are established with response surface methodology. The PEG3350, citrate and sodium chloride concentrations were selected as variables to evaluate the purification impact factors in aqueous two-phase system, including partition coefficients of alpha-amylase, total protein, purification factor and alpha-amylase yield. An experimental space with two-fold purification and over 90% yield of alpha-amylase is achieved through the optimized condition basing on the model. Two systems with low viscosity within said space were further selected to perform alpha-amylase purification and the experimental results coincide well with the calculation of the models, which indicates that the model provides a promising tool for experimental design of protein purification by aqueous two-phase system.

Reproducible preparation and effective separation of PEGylated recombinant human granulocyte colony-stimulating factor with novel "PEG-pellet" PEGylation mode and ion-exchange chromatography.

A novel preparation for polyethylene glycol (PEG) derivatives and chromatographic separation procedure of the PEGylated recombinant human granulocyte colony-stimulating factor (rhG-CSF) were designed to evaluate the reproducibility and scalability at large laboratory-scale level. The new "PEG-pellet" PEGylation mode was successfully applied to control the pH fluctuation during the conjugation reaction, a general problem in traditional liquid-phase conjugation mode. Moreover, two consecutive ion-exchange chromatography steps were successfully used to separate and purify the PEGylated rhG-CSF. Cation-exchange chromatography was firstly applied to separate PEGylated rhG-CSF from intact rhG-CSF, followed by anion-exchange chromatography to obtain individual PEG-rhG-CSF species (mono-, di- and tri-PEGylated rhG-CSF) and remove the excess free PEG. Furthermore, the molecular weight of individual PEGylated rhG-CSF was identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and SDS-PAGE, and cell proliferation activity in vitro was detected by MTT assay using NFS-60 cell.

Ion-exchange chromatography of hepatitis B virus surface antigen from a recombinant Chinese hamster ovary cell line.

About 10% of the Chinese population are chronic carriers of hepatitis B virus (HBV). Thus, the development of a highly efficient process for the preparation of a vaccine based on a recombinant hepatitis B surface antigen (HBsAg) is very important to the Chinese national immunization program. To this end, the ion exchange chromatography recovery of CHO-HBsAg from a recombinant Chinese hamster ovary cell line was shown to increase from about 55 to 80% by the addition of 1% poly(ethylene glycol) (PEG 10,000) to the mobile phase. Furthermore, based on analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), the intact glycoprotein form of CHO-HBsAg was completely preserved by the addition of PEG. In the absence of PEG the glycoprotein form of CHO-HBsAg was also spread out into the high salt elution fraction. High-performance size-exclusion chromatography with on-line multiangle-laser-light scattering (HPSEC-MALLS) analysis was performed to monitor the status of the CHO-HBsAg aggregate structure assembly, particle size and molecular weight distribution after each purification step, and the results showed further that the presence of PEG facilitated the separation and recovery of intact glycoprotein form of CHO-HBsAg and promoted their assembly to proper virus-like particles, which are both important features and prerequisites of their immunogenicity.

Endosomal pH responsive polymers for efficient cancer targeted gene therapy.

Treatment of human diseases at gene level is always limited by effective gene delivery vectors. In this study, we designed and developed an endosomal pH sensitive targeted gene delivery system, folic acid functionalized Schiff-base linked imidazole chitosan (FA-SLICS), for cancer therapy. The FA-SLICS is able to self-assemble plasmid DNA (pDNA) into nano-scaled polyplexes under a neutral condition and to release the loaded pDNA in the endosomal microenvironment due to the presence of pH sensitive Schiff-base moieties along chitosan backbones. The FA-SLICS has negligible cytotoxicity to normal cells (CHO), but displays slight toxicity to cancer cells (HeLa and HepG2). In addition, FA-SLICS can selectively and efficiently transfect FR (folate receptor) positive cells (HeLa cells) as a gene carrier. Therefore, the FA-SLICS should be a promising delivery vector in cancer gene therapy based on its cell targeting capability and intracellular microenvironment controlled delivery mechanism.

Intracellular microenvironment-responsive label-free autofluorescent nanogels for traceable gene delivery.

Gene therapy presents a unique opportunity for the treatment of genetic diseases, but the lack of multifunctional delivery systems has hindered its clinical applications. Here, a new delivery vector, autofluorescent polyethyleneimine (PEI) nanogels, for highly efficient and traceable gene delivery is developed. Different from commercial high-molecular-weight PEI, the cationic nanogels are noncytotoxic and able to be fragmented due to their unique intracellular microenvironment-responsive structures. The biodegradable nanogels can effectively load plasmid DNA (pDNA), and the self-assembled polyplexes can be cleaved after cellular uptake to improve gene transfection efficiency. Most importantly, the nanogels and the nanogel/pDNA polyplexes are autofluorescent. The fluorescence is stable in blood plasma and responsive to the intracellular microenvironment. The breakup of the nanogels or polyplexes leads to the loss of fluorescence, and thus the gene delivery and carrier biodegradation processes can be monitored. The reported multifunctional system demonstrates excellent biocompatibility, high transfection efficiency, responsive biodegradability, controlled gene release, label-free and simultaneous fluorescence tracking, which will provide a new platform for future scientific investigation and practical implications in gene therapy.

Polyethylene glycol improves conjugation of bovine hemoglobin and human serum albumin in a controlled ratio.

Direct conjugation of bovine hemoglobin (Hb) and human serum albumin (HSA) with glutaraldehyde would result in a complex mixture of dimers Hb-Hb, Hb-HSA, HSA-HSA and other oligomers. To obtain a high yield of target Hb-HSA, modulation of the reaction environment was carried out. It was found that polyethylene glycol (PEG), a hydrophilic polymer, could improve the yield of Hb-HSA conjugate. The degree of improvement depended on the molecular weight and concentration of PEG. Under optimum condition of 9% (w/v) of PEG 4000, the reaction proceeded in a controlled mode with conversion yield of starting proteins to Hb-HSA increasing from 6% to 30%. The purity was about 88% of the total conjugates. Furthermore, the impurities were mainly tetrameric molecules of two Hb-HSA conjugates. The improvement could be attributed to the "micro-compartment" created by addition of polyethylene glycol, which brings HSA and Hb close together, thus increasing the chance of conjugation between the two molecules.

[Separation and purification of PEGylated rhG-CSF by two-step ion-exchange chromatography].

In order to separate and purify the PEGylated recombinant human granulocyte stimulating factor (rhG-CSF) at large laboratory-scale level, a two-step ion-exchange chromatographic separation procedure was designed. Cation-exchange chromatography was applied first to separate PEGylated rhG-CSF from un-reacted rhG-CSF, followed by anion-exchange chromatography to dissolve individual PEG-rhG-CSF species (mono-, di- and tri-PEGylated rhG-CSF) and remove the free PEG. The molecular weight of individual PEGylated rhG-CSF was determined by MALDI-TOF and SDS-PAGE. MALDI-TOF mass spectrometry revealed that the molecular weights of mono-, di- and tri-PEGylated rhG-CSF are 23.8 kD, 28.6kD and 33.8kD, respectively. Cell proliferation activity was detected by MTT assay using NFS-60 cell. The in vitro residual bioactivity of mono-, di- and tri-PEGylated rhG-CSF were 90%, 75% and 43% respectively, comparing with the un-conjugated rhG-CSF. These results indicated that the un-conjugated rhG-CSF and excess free PEG can be removed completely and the three conjugate species can be purified into homogeneity by the two consecutive ion-exchange chromatographic steps. The purification procedure is easy to scale-up, high in performance and recovery.

Novel polyethylene glycol derivative suitable for the preparation of mono-PEGylated protein.

A novel methoxypolyethylene glycol (mPEG) derivative, containing a reactive group of 1-methyl pyridinium toluene-4-sulfonate, was synthesized and characterized. The mPEG derivative was successfully conjugated with two proteins: recombinant human granulocyte-colony stimulating factor (rhG-CSF) and consensus interferon (C-IFN). Homogeneous mono-PEGylated proteins were obtained which were identified by high performance size-exclusion chromatography and MALDI-TOF mass spectrometry. The biological activities of the mono-PEGylated rhG-CSF and the mono-PEGylated C-IFN were maintained at 90% and 88%, respectively.

[Polyethylene glycol-accompanied ion-exchange chromatography to purify recombinant hepatitis B virus surface antigen].

The dissociation of virus-like particles of Hepatitis B surface antigen (HBsAg) during the adsorption-desorption on the solid-phase of chromatography is a main challenge for its purification. Herein, poly (ethylene glycol) (PEG) was applied as an additive during the purification of HBsAg from recombinant Chinese hamster ovary (CHO) cell culture to improve the HBsAg recovery and protect its structural assembly. The presence of 1% of PEG10000 in the mobile phase of ion-exchange chromatography (IEC) of DEAE-Sepharose FF column could increase the recovery of HBsAg from about 55% to 80%, with a similar purification (-fold) (about 12) compared with the absence of PEG. Importantly, glycosylated protein forms of HBsAg were reserved well by PEG-accompanied chromatography. Furthermore, size exclusion chromatography-multiangle laser light scattering (SEC-MALLS) analysis was performed on line to monitor the aggregates, particle size and molecular weight distribution of HBsAg. The results demonstrated that the HBsAg particle size and assembly are more homogenous after adding PEG in the mobile phase of IEC than no PEG added in the mobile phase.

Partial purification of alpha-amylase from culture supernatant of Bacillus subtilis in aqueous two-phase systems.

A study was made of the partition and purification of alpha-amylase from a culture supernatant of Bacillus subtilis in the polyethylene glycol (PEG)--citrate aqueous two-phase system (ATPS). Factors that influenced the partition of the protein in this system, including the molecular weight of the PEG, the tie line length of ATPS, the pH value and the sodium chloride concentration, were investigated. Purification of alpha-amylase was attained with a purification factor (PF) of 1.8 and 90% yield at pH 6.0 in a PEG1000-citrate ATPS with short tie line length. By utilizing the salt-out effect of neutral salt, the purification of alpha-amylase was further improved to 2.0 of PF and 80% yield in a PEG3350-citrate ATPS with 4% sodium chloride.