Anti-angiogenic biomolecules in neovascular age-related macular degeneration; therapeutics and drug delivery systems.

Blindness in the elderly is often caused by age-related macular degeneration (AMD). The advanced type of AMD known as neovascular AMD (nAMD) has been linked to being the predominant cause of visual impairment in these people. Multiple neovascular structures including choroidal neovascular (CNV) membranes, fluid exudation, hemorrhages, and subretinal fibrosis, are diagnostic of nAMD. These pathological alterations ultimately lead to anatomical and visual loss. It is known that vascular endothelial growth factor (VEGF), a type of proangiogenic factor, mediates the pathological process underlying nAMD. Therefore, various therapies have evolved to directly target the disease. In this review article, an attempt has been made to discuss general explanations about this disease, all common treatment methods based on anti-VEGF drugs, and the use of drug delivery systems in the treatment of AMD. Initially, the pathophysiology, angiogenesis, and different types of AMD were described. Then we described current treatments and future treatment prospects for AMD and outlined the advantages and disadvantages of each. In this context, we first examined the types of therapeutic biomolecules and anti-VEGF drugs that are used in the treatment of AMD. These biomolecules include aptamers, monoclonal antibodies, small interfering RNAs, microRNAs, peptides, fusion proteins, nanobodies, and other therapeutic biomolecules. Finally, we described drug delivery systems based on liposomes, nanomicelles, nanoemulsions, nanoparticles, cyclodextrin, dendrimers, and composite vehicles that are used in AMD therapy.

Fabrication and investigation of a pentamerous composite based on calix[4]arene functionalized graphene oxide grafted with silk fibroin, cobalt ferrite, and alginate.

This paper presents a new scaffold made from graphene oxide nanosheets, calix[4]arene supramolecules, silk fibroin proteins, cobalt ferrite nanoparticles, and alginate hydrogel (GO-CX[4]/SF/CoFe2O4/Alg). After preparing the composite, we conducted various analyses to examine its structure. These analyses included FTIR, XRD, SEM, EDS, VSM, DLS, and zeta potential tests. Additionally, we performed tests to evaluate the swelling ratio, rheological properties, and compressive mechanical strength of the material. The biological capability of the composite was tested through biocompatiblity, anticancer, hemolysis, antibacterial anti-biofilm assays. Besides, the rheological properties and swelling behaviour of the composite were studied. The results showed that the scaffold is biocompatible with Hu02 cells and the cell viability percentages of 85.23 %, 82.78 %, and 80.18 % for were acquired for 24, 48, and 72 h, respectively. In contrast, the cell viability percentage of BT549 cancer cells were obtained 65.79 %, 60.45 % and 58.16 % for same period which confirmed notable anticancer activity of the product composite. Moreover, a significant antibacterial growth inhibition against E. coli and S. aureus species highlights its potential as an effective antibacterial agent. Furthermore, the observed minimal hemolytic effect (6.56 %) and strong inhibition of P. aeruginosa biofilm formation with a low OD value (0.24) indicate notable hemocompatibility and antibacterial activity.

Expression, Purification, and Biological Evaluation of XTEN-GCSF in a Neutropenic Rat Model.

Granulocyte colony-stimulating factor (GCSF) stimulates the proliferation of neutrophils but it has low serum half-life. Therefore, the present study was done to investigate the effect of XTENylation on biological activity, pharmacokinetics, and pharmacodynamics of GCSF in a neutropenic rat model. XTEN tag was genetically fused to the N-terminal region of GCSF-encoding gene fragment and subcloned into pET28a expression vector. The cytoplasmic expressed recombinant protein was characterized through intrinsic fluorescence spectroscopy (IFS), dynamic light scattering (DLS), and size exclusion chromatography (SEC). In vitro biological activity of the XTEN-GCSF protein was evaluated on NFS60 cell line. Hematopoietic properties and pharmacokinetics were also investigated in a neutropenic rat model. An approximately 140 kDa recombinant protein was detected on SDS-PAGE. Dynamic light scattering and size exclusion chromatography confirmed the increase in hydrodynamic diameter of GCSF molecule after XTENylation. GCSF derivatives showed efficacy in proliferation of NFS60 cell line among which the XTEN-GCSF represented the lowest EC50 value (100.6 pg/ml). Pharmacokinetic studies on neutropenic rats revealed that XTEN polymer could significantly increase protein serum half-life in comparison with the commercially available GCSF molecules. PEGylated and XTENylated GCSF proteins were more effective in stimulation of neutrophils compared to the GCSF molecule alone. XTENylation of GCSF represented promising results in in vitro and in vivo studies. This approach can be a potential alternative to PEGylation strategies for increasing serum half-life of protein.

Design and fabrication of a magnetic nanobiocomposite based on flaxseed mucilage hydrogel and silk fibroin for biomedical and in-vitro hyperthermia applications.

In this research work, a magnetic nanobiocomposite is designed and presented based on the extraction of flaxseed mucilage hydrogel, silk fibroin (SF), and Fe3O4 magnetic nanoparticles (Fe3O4 MNPs). The physiochemical features of magnetic flaxseed mucilage hydrogel/SF nanobiocomposite are evaluated by FT-IR, EDX, FE-SEM, TEM, XRD, VSM, and TG technical analyses. In addition to chemical characterization, given its natural-based composition, the in-vitro cytotoxicity and hemolysis assays are studied and the results are considerable. Following the use of highest concentration of magnetic flaxseed mucilage hydrogel/SF nanobiocomposite (1.75 mg/mL) and the cell viability percentage of two different cell lines including normal HEK293T cells (95.73%, 96.19%) and breast cancer BT549 cells (87.32%, 86.9%) in 2 and 3 days, it can be inferred that this magnetic nanobiocomposite is biocompatible with HEK293T cells and can inhibit the growth of BT549 cell lines. Besides, observing less than 5% of hemolytic effect can confirm its hemocompatibility. Furthermore, the high specific absorption rate value (107.8 W/g) at 200 kHz is generated by a determined concentration of this nanobiocomposite (1 mg/mL). According to these biological assays, this magnetic responsive cytocompatible composite can be contemplated as a high-potent substrate for further biomedical applications like magnetic hyperthermia treatment and tissue engineering.

Magnetic carboxymethyl cellulose-silk fibroin hydrogel: A ternary nanobiocomposite exhibiting excellent biological activity and in vitro hyperthermia of cancer therapy.

In this work, a magnetic nanobiocomposite scaffold based on carboxymethylcellulose (CMC) hydrogel, silk fibroin (SF), and magnetite nanoparticles was fabricated. The structural properties of this new magnetic nanobiocomposite were characterized by various analyses such as FT-IR, XRD, EDX, FE-SEM, TGA and VSM. According to the particle size histogram, most of the particles were between 55 and 77 nm and the value of saturation magnetization of this nanobiocomposite was reported 41.65 emu.g- 1. Hemolysis and MTT tests showed that the designed magnetic nanobiocomposite was compatible with the blood. In addition, the viability percentage of HEK293T normal cells did not change significantly, and the proliferation rate of BT549 cancer cells decreased in its vicinity. EC50 values for HEK293T normal cells after 48 h and 72 h were 3958 and 2566, respectively. Also, these values for BT549 cancer cells after 48 h and 72 h were 0.4545 and 0.9967, respectively. The efficiency of fabricated magnetic nanobiocomposite was appraised in a magnetic fluid hyperthermia manner. The specific absorption rate (SAR) of 69 W/g (for the 1 mg/mL sample at 200 kHz) was measured under the alternating magnetic field (AMF).

Magnetized chitosan hydrogel and silk fibroin, reinforced with PVA: a novel nanobiocomposite for biomedical and hyperthermia applications.

Herein, a multifunctional nanobiocomposite was designed for biological application, amongst which hyperthermia cancer therapy application was specifically investigated. This nanobiocomposite was fabricated based on chitosan hydrogel (CS), silk fibroin (SF), water-soluble polymer polyvinyl alcohol (PVA) and iron oxide magnetic nanoparticles (Fe3O4 MNPs). CS and SF as natural compounds were used to improve the biocompatibility, biodegradability, adhesion and cell growth properties of the nanobiocomposite that can prepare this nanocomposite for the other biological applications such as wound healing and tissue engineering. Since the mechanical properties are very important in biological applications, PVA polymer was used to increase the mechanical properties of the prepared nanobiocomposite. All components of this nanobiocomposite have good dispersion in water due to the presence of hydrophilic groups such as NH2, OH, and COOH, which is one of the effective factors in increasing the efficiency of hyperthermia cancer therapy. The structural analyzes of the hybrid nanobiocomposite were determined by FT-IR, XRD, EDX, FE-SEM, TGA and VSM. Biological studies such as MTT and hemolysis testing proved that it is hemocompatible and non-toxic for healthy cells. Furthermore, it can cause the death of cancer cells to some extent (20.23%). The ability of the nanobiocomposites in hyperthermia cancer therapy was evaluated. Also, the results showed that it can be introduced as an excellent candidate for hyperthermia cancer therapy.

Rational Design and Production of Bioactive Analogs of Recombinant Human Keratinocyte Growth Factor (rhKGF) with Reduced Aggregation Propensity.

Recombinant human keratinocyte growth factor (rhKGF) is a highly aggregation-prone therapeutic protein. The present study aimed to reduce aggregation propensity of rhKGF by engineering the aggregation hotspots. Initially, 21 mutants were designed based on the previously-identified aggregation-prone regions (APRs) and then four of them including mutants No. 4 (L91K, I119K), 7 (V13S, L91K), 14 (L91D, I119D), and 21 (A51E) were selected based on molecular dynamics (MD) simulations for further experimental studies. The recombinantly produced rhKGF and mutants were analyzed regarding secondary structure, thermal stability, aggregation propensity, and biological activity. Far-UV CD spectroscopy showed that the mutants have similar secondary structure with rhKGF. A51E mutant showed enhanced stability and decreased monomer loss under heat stress suggesting its reduced aggregation propensity compared to rhKGF. Mutant No. 14 showed higher stability and less aggregation tendency than mutant No. 4 indicating that only mutations decreasing pI of rhKGF are effective in reducing its aggregation tendency. All of the mutants were at least as potent as rhKGF in stimulating proliferation of MCF-7 epithelial cells. Our results identified A51E as an equally potent, more stable, and less aggregation-prone analog of rhKGF which could be a promising alternative drug candidate for the commercially available rhKGF (Palifermin).

Functionalization of chitosan by metformin, nickel metal ions and magnetic nanoparticles as a nanobiocomposite for purification of alkaline phosphatase from hen's egg yolk.

In this study, a novel and green chitosan-metformin/NiCl2/Fe3O4 nanobiocomposite was synthesized and used to purify alkaline phosphatase (ALPs) from hen's egg yolk. For this purpose, after functionalization of the chitosan biopolymer by terephthaloyl chloride-metformin ligand, the coordination with Ni(II) and magnetization process were performed. The structure and properties of the synthesized nanobiocomposite were then evaluated by using analyzes such as FT-IR, EDX, FE-SEM, XRD, TGA and VSM. Purification of ALPs with chitosan-metformin/NiCl2/Fe3O4 nanobiocomposite is a fast, reusable and cost-effective method. By this protocol, 62% purification efficiency was obtained and the synthesized nanobiocomposite was not attached to other proteins in hen's egg yolk. ALPs was obtained approximately in the pure form and the purification process was evaluated using SDS-PAGE. The reusability of nanobiocomposites was evaluated and a slight decrease in adsorption capacity was observed after 4 cycles.

Extension of human GCSF serum half-life by the fusion of albumin binding domain.

Granulocyte colony stimulating factor (GCSF) can decrease mortality of patients undergo chemotherapy through increasing neutrophil counts. Many strategies have been developed to improve its blood circulating time. Albumin binding domain (ABD) was genetically fused to N-terminal end of GCSF encoding sequence and expressed as cytoplasmic inclusion bodies within Escherichia coli. Biological activity of ABD-GCSF protein was assessed by proliferation assay on NFS-60 cells. Physicochemical properties were analyzed through size exclusion chromatography, circular dichroism, intrinsic fluorescence spectroscopy and dynamic light scattering. Pharmacodynamics and pharmacokinetic properties were also investigated in a neutropenic rat model. CD and IFS spectra revealed that ABD fusion to GCSF did not significantly affect the secondary and tertiary structures of the molecule. DLS and SEC results indicated the absence of aggregation formation. EC50 value of the ABD-GCSF in proliferation of NFS-60 cells was 75.76 pg/ml after 72 h in comparison with control GCSF molecules (Filgrastim: 73.1 pg/ml and PEG-Filgrastim: 44.6 pg/ml). Animal studies of ABD-GCSF represented improved serum half-life (9.3 ± 0.7 h) and consequently reduced renal clearance (16.1 ± 1.4 ml/h.kg) in comparison with Filgrastim (1.7 ± 0.1 h). Enhanced neutrophils count following administration of ABD-GCSF was comparable with Filgrastim and weaker than PEG-Filgrastim treated rats. In vitro and in vivo results suggested the ABD fusion as a potential approach for improving GCSF properties.

Decoration of graphene oxide nanosheets with carboxymethylcellulose hydrogel, silk fibroin and magnetic nanoparticles for biomedical and hyperthermia applications.

In this study, an efficient nanobiocomposite based on graphene oxide (GO), carboxymethylcellulose (CMC) hydrogel, silk fibroin (SF), and Fe3O4 nanoparticles was synthesized. For this purpose and in order to provide a suitable scaffold for the nanobiocomposite, GO was functionalized with a CMC hydrogel via covalent bonding. In the next step, SF was added to the synthesized structure to increase biocompatibility and biodegradability. Fe3O4 was added into the structure by an in situ process and the GO-CMC hydrogel/SF/Fe3O4 nanobiocomposite was synthesized. The synthesized structure was evaluated in terms of toxicity and hemocompatibility and finally, it was used in the hyperthermia technique. This nanocomposite did not destroy healthy HEK293T cells after 48 h and 72 h, while it did annihilate BT549 cancer cells. The GO-CMC hydrogel/SF/Fe3O4 nanobiocomposite has EC50 values of 0.01466 and 0.1415 against HEK293T normal cells and BT549 cancer cells, respectively (after 72 h). The nanocomposite has good potential in hyperthermia applications and at a concentration and a frequency of 1 mg mL-1 and 400 kHz it has a SAR of 67.7 W g-1.