Guoqing Pan

Thermo-responsive molecularly imprinted nanogels for specific recognition and controlled release of proteins

Intelligent nanogels which respond to environmental stimuli with on/off characteristics hold great promise in a number of biomedical applications including drug/gene delivery, diagnostics and therapeutics. Here, we report the synthesis and characterization of a novel type of thermo-responsive nanogel built using the molecular imprinting technique for specific recognition and controlled release of proteins. Using lysozyme as the protein template and N-isopropylacrylamide as the major monomer, protein-imprinted spherical nanogel particles were readily prepared via aqueous precipitation polymerization with the aid of a surfactant, sodium dodecyl sulfate (SDS). Simply by adjusting the SDS amount during polymerization, the size of nanogels could be finely controlled, ranging from a few hundred down to a few dozen nanometers. Compared to non-imprinted counterparts, the lysozyme-imprinted nanogels possessed higher rebinding capacity, more rapid rebinding kinetics, and much higher specificity toward lysozyme. Importantly, both the rebinding and release characteristics of lysozyme-imprinted nanogels showed dramatic temperature-dependence, with clear on–off transition around 33 °C, i.e., the volume phase transition temperature of the thermo-responsive polymer poly(N-isopropylacrylamide). Therefore, we have developed a facile yet versatile approach to fabricate molecularly imprinted nanogels of well controlled sizes and thermo-responsive binding/release properties toward specific biomolecules, which may facilitate a broad spectrum of applications ranging from bioseparation and biosensing to drug delivery and therapeutics.

Thermo‐Responsive Hydrogel Layers Imprinted with RGDS Peptide: A System for Harvesting Cell Sheets

Cell sheet technology is a novel approach to preparing and harvesting monolayer cell sheets by using poly(N-isopropylacrylamide)(PNIPAAm)-modified surfaces as thermoresponsive cell culture substrates. At lower temperatures the cultured cells detach spontaneously as the surface of the substrate changes from hydrophobic to hydrophilic. In this way, an intact cell monolayer can be harvested non-invasively together with its underlying extracellular matrix (ECM). As a frequently used way to achieve scaffold-free tissue engineering, cell sheet technology holds great promise in cell-based regenerative medicine. Considering the limited availability of autologous cells and the timeliness requirement of clinical treatments, current attempts at improving the efficiency of thermo-responsive cell sheet harvest systems face a dilemma. On the one hand, the cell culture substrate must be very cell-adhesive to markedly promote rapid adhesion and proliferation of the limited autologous cells for a timely therapy. On the other hand, the same substrate should become very cell-repulsive after the formation of a confluent cell monolayer such that the mature cell sheet can be rapidly released without hurting the cells and their underlying ECM. Specifically, simple regulation of the chemical composition or the topography of the surface can only either promote cell adhesion or accelerate cell detachment and these surfaces commonly have very low bioactivity. Introducing cell-adhesive biomolecules by means of covalent binding or physical adsorption can improve the surface bioactivity but result in the inevitable deceleration of cell detachment and serious leakage of the biomolecules, respectively. Thus far, no single method without additional auxiliary means can effectively enhance cell adhesion during culture as well as facilitate the rapid harvest of cell sheets. To conquer this long-standing problem, we conceive of introducing cell-adhesive biomolecules to a thermo-responsive cell culture substrate in a reversible way and modulating them through temperature-dependent interactions. In this case, biomolecules can be stably immobilized on the substrate at cell culture temperature (37 8C), while they can be released as the temperature drops (e.g., 20 8C), thus facilitating both the initial cell adhesion and the final detachment of the cell sheet. With this strategy in mind, we find that the reversible interaction known as “specific binding” in noncovalent molecular imprinting is very appealing. As is well known, polymeric receptors with tailor-made recognition sites and “specific binding” properties comparable with those of natural receptors can be easily prepared by molecular imprinting. More importantly, molecularly imprinted polymers (MIPs) containing thermo-responsive recognition sites (i.e., the sites with temperature-dependent interactions between MIPs and targeted molecules) can be readily obtained using PNIPAAm-based materials. We herein report a novel system for harvesting cell sheets which relies on a PNIPAAm-based MIP hydrogel layer with thermo-responsive affinity toward specific biomolecules (Scheme 1). The commonly used cell-adhesive peptide ArgGly-Asp-Ser (RGDS) was chosen as the target biomolecule to demonstrate the proof-of-principle of our strategy. In our design, the thermo-responsive recognition sites in the MIPs were the tactic used to achieve temperature-dependent interactions between RGDS molecules and cell culture substrate. Specifically, besides the temperature-induced change of the surface wettability, the thermo-responsive recognition sites on the MIP hydrogel layer also resulted in the stable recognition and binding of RGDS at 37 8C and the triggered release of RGDS as the temperature was decreased. In contrast to the introduction of biomolecules by means of covalent binding or physical absorption, the thermo-responsive affinity of the MIPs toward RGDS not only significantly promotes cell adhesion during cell culture (37 8C) but also facilitates the detachment of cell sheets at low temperature (20 8C). To the best of our knowledge, although MIPs have exhibited some expanded bioapplications with the emergence of various biomolecule (e.g., proteins or peptides) imprinted polymers, this study is the first demonstration of molecular imprinting as a methodology to biofunctionalize thermoresponsive cell culture substrates to harvest cell sheets for potential biomedical applications. To achieve the best affinity between RGDS and the MIP matrix during cell culture, the imprinting process was performed by redox-initiated polymerization at 37 8C in [*] Dr. G. Pan, Q. Guo, Prof. Dr. H. Yang, Prof. Dr. B. Li Department of Orthopaedics The First Affiliated Hospital of Soochow University 188 Shizi Street, Suzhou, Jiangsu 215006 (China) and Orthopaedic Institute, Soochow University 708 Renmin Road, Suzhou, Jiangsu 215007 (China) E-mail: yueer@suda.edu.cn binli@suda.edu.cn

Melatonin reverses H2O2‐induced premature senescence in mesenchymal stem cells via the SIRT1‐dependent pathway

Mesenchymal stem cells (MSCs) represent an attractive source for stem cell‐based regenerative therapy, but they are vulnerable to oxidative stress‐induced premature senescence in pathological conditions. We previously reported antioxidant and antiarthritic effects of melatonin on MSCs against proinflammatory cytokines. In this study, we hypothesized that melatonin could protect MSCs from premature senescence induced by hydrogen peroxide (H2O2) via the silent information regulator type 1 (SIRT1)‐dependent pathway. In response to H2O2 at a sublethal concentration of 200 μm, human bone marrow‐derived MSCs (BM‐MSCs) underwent growth arrest and cellular senescence. Treatment with melatonin before H2O2 exposure cannot significantly prevent premature senescence; however, treatment with melatonin subsequent to H2O2 exposure successfully reversed the senescent phenotypes of BM‐MSCs in a dose‐dependent manner. This result was made evident by improved cell proliferation, decreased senescence‐associated β‐galactosidase activity, and the improved entry of proliferating cells into the S phase. In addition, treatment with 100 μm melatonin restored the osteogenic differentiation potential of BM‐MSCs that was inhibited by H2O2‐induced premature senescence. We also found that melatonin attenuated the H2O2‐stimulated phosphorylation of p38 mitogen‐activated protein kinase, decreased expression of the senescence‐associated protein p16INK4α, and increased SIRT1. Further molecular experiments revealed that luzindole, a nonselective antagonist of melatonin receptors, blocked melatonin‐mediated antisenescence effects. Inhibition of SIRT1 by sirtinol counteracted the protective effects of melatonin, suggesting that melatonin reversed the senescence in cells through the SIRT1‐dependent pathway. Together, these findings lay new ground for understanding oxidative stress‐induced premature senescence and open perspectives for therapeutic applications of melatonin in stem cell‐based regenerative medicine.

Surface biofunctional drug-loaded electrospun fibrous scaffolds for comprehensive repairing hypertrophic scars.

Incorporation of bioactive drugs and biofunctionalization of polyester fibrous scaffolds are essential means to improve their bio-functions and histocompatibility for regenerative medicine. However, it is still a challenge to biofunctionalize such drug carriers via traditional biochemical methods while maintaining their properties without changes in drug activity and loading ratio. Here, we demonstrated a facile approach for biofunctionalization of PLGA fibrous scaffolds with various molecules (i.e., PEG polymer, RGD peptide and bFGF growth factor for cell repellent, adhesion and proliferation, respectively) via mussel-Inspired poly(dopamine) (PDA) coating in aqueous solution. By virtue of the mild and efficient nature of this approach, the drug-loaded PLGA fibers could be easily biofunctionalized and showed negligible effects on the scaffold properties, especially drug activity and loading ratio. Further, in vivo study showed that, a ginsenoside-Rg3-loaded fibrous scaffold functionalized with bFGF growth factor could not only promote the early-stage wound healing in rabbit ear wounds (bio-signal from bFGF), but also inhibit later-stage hypertrophic scars formation (release of Rg3 drug). Therefore, the mussel-inspired method for bio-modification provides a facile and effective strategy to combine drug and bio-function in one system, thus facilitating a synergistic effect of drug-therapy and bio-signal when such biomaterial is used for regenerative medicine.

Optimization of intrinsic and extrinsic tendon healing through controllable water-soluble mitomycin-C release from electrospun fibers by mediating adhesion-related gene expression

To balance intrinsic and extrinsic healing during tendon repair is challenging in tendon surgery. We hypothesized that by mediating apoptotic gene and collagen synthesis of exogenous fibroblasts, the adhesion formation induced by extrinsic healing could be inhibited. With the maintenance of intrinsic healing, the tendon could be healed with proper function with no adhesion. In this study, we loaded hydrophilic mitomycin-C (MMC) into hyaluronan (HA) hydrosols, which were then encapsulated in poly(L-lactic acid) (PLLA) fibers by micro-sol electrospinning. This strategy successfully provided a controlled release of MMC to inhibit adhesion formations with no detrimental effect on intrinsic healing. We found that micro-sol electrospinning was an effective and facile approach to incorporate and control hydrophilic drug release from hydrophobic polyester fibers. MMC exhibited an initially rapid, and gradually steadier release during 40 days, and the release rates could be tuned by its concentration. Inxa0vitro studies revealed that low concentrations of MMC could inhibit fibroblast adhesion and proliferation. When lacerate tendons were healed using the MMC-HA loaded PLLA fibers inxa0vivo, they exhibited comparable mechanical strength to the naturally healed tendons but with no significant presence of adhesion formation. We further identified the up-regulation of apoptotic protein Bax expression and down-regulation of proteins Bcl2, collage I, collagen III and α-SMA during the healing process associated with minimum adhesion formations. This approach presented here leverages new advances in drug delivery and nanotechnology and offers a promising strategy to balance intrinsic and extrinsic tendon healing through modulating genes associated with fibroblast apoptosis and collagen synthesis.

A Hierarchical Porous Bowl-like PLA@MSNs-COOH Composite for pH-Dominated Long-Term Controlled Release of Doxorubicin and Integrated Nanoparticle for Potential Second Treatment

We chemically integrated mesoporous silica nanoparticles (MSNs) and macroporous bowl-like polylactic acid (pBPLA) matrix, for noninvasive electrostatic loading and long-term controlled doxorubicin (DOX) release, to prepare a hierarchical porous bowl-like pBPLA@MSNs-COOH composite with a nonspherical and hierarchical porous structure. Strong electrostatic interaction with DOX rendered excellent encapsulation efficiency (up to 90.14%) to the composite. DOX release showed pH-dominated drug release kinetics; thus, maintaining a weak acidic pH (e.g., 5.0) triggered sustained release, suggesting the composites great potential for long-term therapeutic approaches. In-vitro cell viability assays further confirmed that the composite was biocompatible and that the loaded drugs were pharmacologically active, exhibiting dosage-dependent cytotoxicity. Additionally, a wound-healing assay revealed the composites intrinsic ability to inhibit cell migration. Moreover, pH- and time-dependent leaching of the integrated MSNs due to pBPLA matrix degradation allow us to infer that the leached (and drug loaded) MSNs may be engulfed by cancer cells contributing to a second wave of DOX-mediated cytotoxicity following pH-triggered DOX release.

Self-coated interfacial layer at organic/inorganic phase for temporally controlling dual-drug delivery from electrospun fibers.

Implantable tissue engineering scaffolds with temporally programmable multi-drug release are recognized as promising tools to improve therapeutic effects. A good example would be one that exhibits initial anti-inflammatory and long-term anti-tumor activities after tumor resection. In this study, a new strategy for self-coated interfacial layer on drug-loaded mesoporous silica nanoparticles (MSNs) based on mussel-mimetic catecholamine polymer (polydopamine, PDA) layer was developed between inorganic and organic matrix for controlling drug release. When the interface PDA coated MSNs were encapsulated in electrospun poly(L-lactide) (PLLA) fibers, the release rates of drugs located inside/outside the interfacial layer could be finely controlled, with short-term release of anti-inflammation ibuprofen (IBU) for 30 days in absence of interfacial interactions and sustained long-term release of doxorubicin (DOX) for 90 days in presence of interfacial interactions to inhibit potential tumor recurrence. The DOX@MSN-PDA/IBU/PLLA hybrid fibrous scaffolds were further found to inhibit proliferation of inflammatory macrophages and cancerous HeLa cells, while supporting the normal stromal fibroblast adhesion and proliferation at different release stages. These results have suggested that the interfacial obstruction layer at the organic/inorganic phase was able to control the release of drugs inside (slow)/outside (rapid) the interfacial layer in a programmable manner. We believe such interface polymer strategy will find applications in where temporally controlled multi-drug delivery is needed.

Down-regulating ERK1/2 and SMAD2/3 phosphorylation by physical barrier of celecoxib-loaded electrospun fibrous membranes prevents tendon adhesions.

Peritendinous adhesions, as a major problem in hand surgery, may be due to the proliferation of fibroblasts and excessive collagen synthesis, in which ERK1/2 and SMAD2/3 plays crucial roles. In this study, we hypothesized that the complication progression could be inhibited by down-regulating ERK1/2 and SMAD2/3 phosphorylation of exogenous fibroblasts with celecoxib. Celecoxib was incorporated in poly(l-lactic acid)-polyethylene glycol (PELA) diblock copolymer fibrous membranes via electrospinning. Results of an in vitro drug release study showed celecoxib-loaded membrane had excellent continuous drug release capability. It was found that celecoxib-loaded PELA membranes were not favorable for the rabbit fibroblast and tenocyte adhesion and proliferation. In a rabbit tendon repair model, we first identified ERK1/2 and SMAD2/3 phosphorylation as a critical driver of early adhesion formation progression. Celecoxib released from PELA membrane was found to down-regulate ERK1/2 and SMAD2/3 phosphorylation, leading to reduced collagen I and collagen Ⅲ expression, inflammation reaction, and fibroblast proliferation. Importantly, the celecoxib-loaded PELA membranes successfully prevented tissue adhesion compared with control treatment and unloaded membranes treatment. This approach offers a novel barrier strategy to block tendon adhesion through targeted down-regulating of ERK1/2 and SMAD2/3 phosphorylation directly within peritendinous adhesion tissue.

Doxorubicin-loaded mesoporous silica nanoparticle composite nanofibers for long-term adjustments of tumor apoptosis.

There is a high local recurrence (LR) rate in breast-conserving therapy (BCT) and enhancement of the local treatment is promising as a way to improve this. Thus we propose a drug delivery system using doxorubicin (DOX)-loaded mesoporous silica nanoparticle composite nanofibers which can release anti-tumor drugs in two phases-burst release in the early stage and sustained release at a later stage-to reduce the LR of BCT. In the present study, we designed a novel composite nanofibrous scaffold to realize the efficient release of drugs by loading both DOX and DOX-loaded mesoporous silica nanoparticles into an electrospun PLLA nanofibrous scaffold. In vitro results demonstrated that this kind of nanomaterial can release DOX in two phases, and the results of in vivo experiments showed that this hybrid nanomaterial significantly inhibited the tumor growth in a solid tumor model. Histopathological examination demonstrated that the apoptosis of tumor cells in the treated group over a 10 week period was significant. The anti-cancer effects were also accompanied with decreased expression of Bcl-2 and TNF-α, along with up-regulation of Bax, Fas and the activation of caspase-3 levels. The present study illustrates that the mesoporous silica nanoparticle composite nanofibrous scaffold could have anti-tumor properties and could be further developed as adjuvant therapeutic protocols for the treatment of cancer.

Tumor‐Triggered Controlled Drug Release from Electrospun Fibers Using Inorganic Caps for Inhibiting Cancer Relapse

A smart, tumor-trigged, controlled drug release using inorganic caps with CO3 (2-) functional groups in electrospun fibers is presented for inhibiting cancer relapse. When the drug-loaded intelligent electrospun fibers encounter pathological acidic environments, the inorganic gates react with the acids and produce CO2 gas, which enables water penetration into the core of the fibers to induce rapid drug release.