In vitro combinatorial anticancer effects of 5-fluorouracil and curcumin loaded N,O-carboxymethyl chitosan nanoparticles toward colon cancer and in vivo pharmacokinetic studies.

Colon cancer is the third most leading causes of death due to cancer worldwide and the chemo drug 5-fluorouracil's (5-FU) applicability is limited due to its non-specificity, low bioavailability and overdose. The efficacy of 5-FU in colon cancer chemo treatment could be improved by nanoencapsulation and combinatorial approach. In the present study curcumin (CUR), a known anticancer phytochemical, was used in combination with 5-FU and the work focuses on the development of a combinatorial nanomedicine based on 5-FU and CUR in N,O-carboxymethyl chitosan nanoparticles (N,O-CMC NPs). The developed 5-FU-N,O-CMC NPs and CUR-N,O-CMC NPs were found to be blood compatible. The in vitro drug release profile in pH 4.5 and 7.4 showed a sustained release profile over a period of 4 days. The combined exposure of the nanoformulations in colon cancer cells (HT 29) proved the enhanced anticancer effects. In addition, the in vivo pharmacokinetic data in mouse model revealed the improved plasma concentrations of 5-FU and CUR which prolonged up to 72 h unlike the bare drugs. In conclusion, the 5-FU and CUR released from the N,O-CMC NPs produced enhanced anticancer effects in vitro and improved plasma concentrations under in vivo conditions.

Fabrication and characterization of chitosan/gelatin/nSiO2 composite scaffold for bone tissue engineering.

A 3D nanocomposite scaffold of chitosan, gelatin and nano-silica was fabricated by lyophilization to test the hypothesis that incorporation of nano-SiO2 could produce a better candidate for bone tissue engineering compared to pure chitosan and chitosan/gelatin scaffolds. The prepared scaffold was characterized using SEM and FTIR. Porosity, density, swelling, degradation, mechanical integrity, biomineralization and protein adsorption studies, favored it in comparison to the conventional chitosan and chitosan/gelatin scaffolds. In vitro cyto-compatablity, cell attachment-proliferation, ALP activity studies performed using MG-63 cells, advocate its remarkable performance. These cumulative results indicate the prepared nanocomposite scaffold as a prospective candidate for bone tissue engineering.

Fabrication and characterization of multiscale electrospun scaffolds for cartilage regeneration.

Recently, scaffolds for tissue regeneration purposes have been observed to utilize nanoscale features in an effort to reap the cellular benefits of scaffold features resembling extracellular matrix (ECM) components. However, one complication surrounding electrospun nanofibers is limited cellular infiltration. One method to ameliorate this negative effect is by incorporating nanofibers into microfibrous scaffolds. This study shows that it is feasible to fabricate electrospun scaffolds containing two differently scaled fibers interspersed evenly throughout the entire construct as well as scaffolds containing fibers composed of two discrete materials, specifically fibrin and poly(ε-caprolactone). In order to accomplish this, multiscale fibrous scaffolds of different compositions were generated using a dual extrusion electrospinning setup with a rotating mandrel. These scaffolds were then characterized for fiber diameter, porosity and pore size and seeded with human mesenchymal stem cells to assess the influence of scaffold architecture and composition on cellular responses as determined by cellularity, histology and glycosaminoglycan (GAG) content. Analysis revealed that nanofibers within a microfiber mesh function to maintain scaffold cellularity under serum-free conditions as well as aid the deposition of GAGs. This supports the hypothesis that scaffolds with constituents more closely resembling native ECM components may be beneficial for cartilage regeneration.

In vitro and in vivo evaluation of microporous chitosan hydrogel/nanofibrin composite bandage for skin tissue regeneration.

In this work, we have developed chitosan hydrogel/nanofibrin composite bandages (CFBs) and characterized using Fourier transform-infrared spectroscopy and scanning electron microscopy. The homogeneous distribution of nanofibrin in the prepared chitosan hydrogel matrix was confirmed by phosphotungstic acid-hematoxylin staining. The mechanical strength, swelling, biodegradation, porosity, whole-blood clotting, and platelet activation studies were carried out. In addition, the cell viability, cell attachment, and infiltration of the prepared CFBs were evaluated using human umbilical vein endothelial cells (HUVECs) and human dermal fibroblast (HDF) cells. It was found that the CFBs were microporous, flexible, biodegradable, and showed enhanced blood clotting and platelet activity compared to the one without nanofibrin. The prepared CFBs were capable of absorbing fluid and this was confirmed when immersed in phosphate buffered saline. Cell viability studies on HUVECs and HDF cells proved the nontoxic nature of the CFBs. Cell attachment and infiltration studies showed that the cells were found attached and proliferated on the CFBs. In vivo experiments were carried out in Sprague-Dawley rats and found that the wound healing occurred within 2 weeks when treated with CFBs than compared to the bare wound and wound treated with Kaltostat. The deposition of collagen was found to be more on CFB-treated wounds compared to the control. The above results proved the use of these CFBs as an ideal candidate for skin tissue regeneration and wound healing.

Fabrication of electrospun poly (lactide-co-glycolide)-fibrin multiscale scaffold for myocardial regeneration in vitro.

Myocardial tissue engineering is one of the most promising treatment strategies to restore heart function after a massive heart attack. The biomaterials, cells, and scaffold design play important roles in engineering of heart tissue. In this study, we have developed a fibrin-based multiscale electrospun composite scaffold for myocardial regeneration. Fibrin is the natural wound-healing matrix having angiogenic potential and comprehensively used for tissue engineering applications. It provides a natural environment for cell attachment, migration, and proliferation. Morphological, chemical, and mechanical characterization of the scaffolds was done by scanning electron microscopy, fibrin-specific phosphotungstic acid hematoxylin staining, and mechanical testing. The fiber diameters of fibrin nanofibers range from 50 to 300 nm and that of poly (lactide-co-glycolide) microfibers range from 2 to 4 µm, which mimics the structural hierarchy of native myocardial tissue. Our results indicate that this scaffold enhances the differentiation of mesenchymal stem cells into cardiomyocytes. The cardiac phenotype of the cells was confirmed by the presence of cardiac-specific proteins like α-sarcomeric actinin, troponin, tropomyosin, desmin, and atrial natriuretic peptide Estimation of D-Dimer in the culture supernatant for 2 weeks and analysis of scaffold for 3 weeks of in vitro culture of cardiomyocytes indicated the degradation of fibrin and presence of newly synthesized collagen respectively. Our results demonstrate the promising potential of this scaffold for myocardial tissue engineering applications.

In vitro evaluation of electrospun PCL/nanoclay composite scaffold for bone tissue engineering.

Polycaprolactone (PCL) is a widely accepted synthetic biodegradable polymer for tissue engineering, however its use in hard tissue engineering is limited because of its inadequate mechanical strength and low bioactivity. In this study, we used halloysite nanoclay (NC) as an inorganic filler material to prepare PCL/NC fibrous scaffolds via electrospinning technique after intercalating NC within PCL by solution intercalation method. The obtained nanofibrous mat was found to be mechanically superior to PCL fibrous scaffolds. These scaffolds allowed greater protein adsorption and enhanced mineralization when incubated in simulated body fluid. Moreover, our results indicated that human mesenchymal stem cells (hMSCs) seeded on these scaffolds were viable and could proliferate faster than in PCL scaffolds as confirmed by fluorescence and scanning electron microscopic observations. Further, osteogenic differentiation of hMSCs on nanoclay embedded scaffolds was demonstrated by an increase in alkaline phosphatase activity when compared to PCL scaffold without nanoclay. All of these results suggest the potential of PCL/NC scaffolds for bone tissue engineering.

Synthesis and characterization of chitosan/chondroitin sulfate/nano-SiO2 composite scaffold for bone tissue engineering.

Chitosan, a natural polymer, is a biomaterial which is known to be osteoconductive but lacking in mechanical strength. In this work, to further enhance the mechanical property and biocompatibility of chitosan, we combined it with both chondroitin sulfate, a natural glycosaminoglycan found in bone, and nano-SiO2. The composite scaffold of chitosan/chondroitin sulfate/nano-SiO2 was fabricated by lyophilization. The nanocomposite scaffold showed enhanced porosity, degradation, mechanical integrity, biomineralization and protein adsorption. Biocompatibility and cell attachment-proliferation studies performed using MG-63 cells, advocate its better performance in vitro. To improve the cell seeding efficiency, we coated the scaffold surface with fibrin, which enhanced the initial cell attachment. The cumulative results suggest this novel nanocomposite scaffold to be a suitable candidate for bone tissue engineering.

Fibrin nanoconstructs: a novel processing method and their use as controlled delivery agents.

Fibrin nanoconstructs (FNCs) were prepared through a modified water-in-oil emulsification-diffusion route without the use of any surfactants, resulting in a high yield synthesis of fibrin nanotubes (FNTs) and fibrin nanoparticles (FNPs). The fibrin nanoconstructs formed an aligned structure with self-assembled nanotubes with closed heads that eventually formed spherical nanoparticles of size ~250 nm. The nanotubes were typically ~700 nm long and 150-300 nm in diameter, with a wall thickness of ~50 nm and pore diameter of about 150-250 nm. These constructs showed high stability against aggregation indicated by a zeta potential of -44 mV and an excellent temperature stability upto 200 °C. Furthermore, they were found to be enzymatically degradable, thereby precluding any long term toxicity effects. These unique fibrin nanostructures were analyzed for their ability to deliver tacrolimus, an immunosuppressive drug that is used widely to prevent the initial phase of tissue rejection during allogenic transplantation surgeries. Upon conjugation with tacrolimus, a drug encapsulation efficiency of 66% was achieved, with the in vitro release studies in PBS depicting a sustained and complete drug release over a period of one week at the physiological pH of 7.4. At a more acidic pH, the drug release was very slow, suggesting their potential for oral-intestinal drug administration as well. The in vivo drug absorption rates analyzed in Sprague Dawley rats further confirmed the sustained release pattern of tacrolimus for both oral and parenteral delivery routes. The novel fibrin nanoconstructs developed using a green chemistry approach thus proved to be excellent biodegradable nanocarriers for oral as well as parenteral administrations, with remarkable potential also for delivering specific growth factors in tissue engineering scaffolds.

A novel method for the fabrication of fibrin-based electrospun nanofibrous scaffold for tissue-engineering applications.

In this study, fibrin, which is superior to fibrinogen in both structural and functional properties, has for the first time been electrospun successfully into uniform nano fibers resembling the extracellular matrix (ECM). The methods of fabrication and characterization of this unique scaffold are presented. Using poly (vinyl) alcohol as an "electrospinning-driving" polymer, we have developed a novel method for the fabrication of fibrin into a nanofibrous scaffold for various tissue-engineering applications starting from human-plasma-derived fibrinogen and thrombin and combining these ingredients within the syringe of an electrospinning setup under high voltage. In this fashion, fibrin nanofibrous scaffold is produced in a one-step approach without the need for subsequent cross-linking by synthetic agents that compromise the biological properties of the scaffold. Characterization of the electrospun membrane was done by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, and fibrin specific phosphotungstic acid hematoxylin staining. SEM data revealed the formation of bead-free fibers with a dimension ranging from 50-500 nm, which exactly mimics the fiber diameter of native ECM. Cell attachment and proliferation studies revealed that the scaffold supports the attachment, spreading, and proliferation of human umbilical cord blood-derived mesenchymal stem cells.

Multifunctional chitin nanogels for simultaneous drug delivery, bioimaging, and biosensing.

In this work, we developed biodegradable chitin nanogels (CNGs) by controlled regeneration method. For multifunctionalization, we have conjugated CNGs with MPA-capped-CdTe-QDs (QD-CNGs) for the in vitro cellular localization studies. In addition, the Bovine Serum Albumin (BSA) was loaded on to QD-CNGs (BSA-QD-CNGs). The CNGs, QD-CNGs, and BSA-QD-CNGs were well-characterized by SEM and AFM, which shows that the nanogels are in the range of <100 nm. These were further characterized by FT-IR and Cyclic Voltametry. The cytocompatibility assay showed that the nanogels are nontoxic to L929, NIH-3T3, KB, MCF-7, PC3, and VERO cells. The cell uptake studies of the QD-CNGs were analyzed, which showed retention of these nanogels inside the cells (L929, PC3, and VERO). In addition, the protein loading efficiency of the nano gels has also been analyzed. Our preliminary studies reveal that these multifunctionalized nanogels could be useful for drug delivery with simultaneous imaging and biosensing.

Chitin scaffolds in tissue engineering.

Tissue engineering/regeneration is based on the hypothesis that healthy stem/progenitor cells either recruited or delivered to an injured site, can eventually regenerate lost or damaged tissue. Most of the researchers working in tissue engineering and regenerative technology attempt to create tissue replacements by culturing cells onto synthetic porous three-dimensional polymeric scaffolds, which is currently regarded as an ideal approach to enhance functional tissue regeneration by creating and maintaining channels that facilitate progenitor cell migration, proliferation and differentiation. The requirements that must be satisfied by such scaffolds include providing a space with the proper size, shape and porosity for tissue development and permitting cells from the surrounding tissue to migrate into the matrix. Recently, chitin scaffolds have been widely used in tissue engineering due to their non-toxic, biodegradable and biocompatible nature. The advantage of chitin as a tissue engineering biomaterial lies in that it can be easily processed into gel and scaffold forms for a variety of biomedical applications. Moreover, chitin has been shown to enhance some biological activities such as immunological, antibacterial, drug delivery and have been shown to promote better healing at a faster rate and exhibit greater compatibility with humans. This review provides an overview of the current status of tissue engineering/regenerative medicine research using chitin scaffolds for bone, cartilage and wound healing applications. We also outline the key challenges in this field and the most likely directions for future development and we hope that this review will be helpful to the researchers working in the field of tissue engineering and regenerative medicine.