Composite Hydrogels of Ultrasound-Assisted-Digested Formic Acid-Decellularized Extracellular Matrix and Sacchachitin Nanofibers Incorporated with Platelet-Rich Plasma for Diabetic Wound Treatment.

In this study, an ultrasound-assisted digestion method of a formic acid-decellularized extracellular matrix (dECM) of porcine skin was developed and optimized to form UdECM hydrogels for diabetic wound healing. Results demonstrated that ultrasonication improved the extraction rate of collagen from dECM samples, preserved the collagen content of dECM, reduced residual cells, and extracted greater DNA contents. Scanning electron microscope (SEM) analyses were performed, which demonstrated the optimal porosity on the surface and density of the cross-section in the hydrogel structure, which could control the release of growth factors embedded in UdECM hydrogels at desirable rates to boost wound healing. A wound-healing study was conducted with six different composite hydrogels, both empty materials and materials enriched with rat platelet-rich plasma (R-PRP), sacchachitin nanofibers (SCNFs), and TEMPO-oxidized sacchachitin in diabetic rats. The assessment based on scars stained with hematoxylin and eosin (H&E), Masson's trichrome (MT), and a cluster of differentiation 31 (CD31) staining showed that the UdECM/SC/R-PRP treatment group had the most significant efficacy of promoting healing and even recovery of diabetic wounds to normal tissues. UdECM/R-PRP and UdECM/SCNFs demonstrated better healing rates than UdECM hydrogel scaffolds, which had only recovered 50% resemblance to normal skin. Treatment with both UdECM/TEMPO 050 and UdECM/TEMPO 050/R-PRP hydrogel scaffolds was ranked last, with even poorer efficacy than UdECM hydrogels. In summary, formulated UdECM and SCNF hydrogels loaded with PRP showed synergistic effects of accelerating wound healing and ultimately stimulating the wound to recover as functional tissues. This newly UdECM/SCNF composite hydrogel has promising potential for healing and regenerating diabetic wounds.

Poloxamer sols endowed with in-situ gelability and mucoadhesion by adding hypromellose and hyaluronan for prolonging corneal retention and drug delivery.

The purpose of this study was to develop poloxamer (P407)-based in-situ thermogellable hydrogels with reducing concentration of P407 by adding hypromellose (HPMC) and with enhancing mucoadhesion of resulting hydrogels by adding hyaluronic acid (HA) for prolonging ocular delivery of hydroxypropyl-ß-cyclodextrin (HPßCD)-solubilized testosterone (TES). Results demonstrated that 0.5% TES solution was successfully solubilized with adding 10% HPßCD. Non-gellable 13% P407 sol became in-situ gellable with adding 2.0-2.5% HPMC and mucoadhesibility was further imporved with adding 0.3% HA-L (low MW) or HA-H (high MW). Optimized 0.5% HPßCD-solubilized TES P407-based thermogellable hydrogels with enhancement of mucoadhesion for prolonging ocular delivery comprised 13% P407, 2.5% HPMC, and 0.3% HA-L or HA-H. Furthermore, rheological measurements under simulated eye blinking confirmed that non-thixotropic properties of optimized hydrogels could be spreaded evenly and retain a greater amount of drug-loaded hydrogels on the ocular surface for a longer period to prolong drug delivery. Compared with conventional eye drops, the prolonged residence time of optimized hydrogels from ex vivo and in vivo studies were observed, indicating relationships between rheological properties and in vivo performances. It was concluded that P407-based thermosensitive hydrogels with reducing concentration of P407 and enhancing mucoadhesion was successfully formulated by adding 2.5% HPMC and 0.3% HA in 13% P407 for potentially accomplishing effective clinical treatment of DED.

Combined Docetaxel/Pictilisib-Loaded mPEGylated Nanocarriers with Dual HER2 Targeting Antibodies for Synergistic Chemotherapy of Breast Cancer.

Introduction: Approximately 15%~30% of breast cancers have gene amplification or overexpression of the human epidermal growth factor receptor 2 (HER2), resulting in the chemotherapy resistance, a more-aggressive phenotype and poor prognosis. Methods: We propose a strategy of nanocarriers co-loaded with docetaxel (DTX) and pictilisib (PIC) at a synergistic ratio and non-covalently bound with dual anti-HER2 epitopes bispecific antibodies (BsAbs: anti-HER2-IV/methoxy-polyethylene glycol (mPEG) and anti-HER2-II/methoxy-PEG) for synergistic targeting to overcome the therapeutic dilemmas of the resistance for HER2-targetable chemodrugs. DTX/PIC-loaded nanocarriers (D/P_NCs) were prepared with single emulsion methods and characterized using dynamic light scattering analysis, and the drug content was assayed by high-performance liquid chromatographic method. The integrity and function of BsABs were evaluated using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA). The in vitro cell studies and in vivo breast tumor-bearing mice model were used to evaluate the anti-cancer effect and biosafety of formulations. Results: D/P_NCs optimally prepared exhibited a spherical morphology with small particle sizes (~140 nm), high drug loading (~5.5%), and good colloidal stability. The synergistic tumor cytotoxicity of loading DTX and PIC at 2:1 ratio in D/P_NCs was discovered. The BsAbs are successfully decorated on mPEGylated DTX/PIC-loaded nanocarriers via anti-mPEG moiety. In vitro studies revealed that non-covalent decoration with dual BsAbs on D_P-NCs significantly and synergistically increased cellular uptake, while with loading DTX and PIC at a synergistic ratio of 2:1 in D/P_NCs further resulted in synergistic cytotoxicity. In vivo tumor inhibition studies showed the comparable results for synergistic antitumor efficacy while minimizing systemic toxicity of chemodrugs. Conclusion: Non-covalent modification with dual distinct epitopes BsAbs on the nanocarriers loaded with dual chemodrugs at a synergistic ratio was expected to be a promising therapeutic platform to overcome the chemoresistance of various cancers and warrants further development for future therapy in the clinical.

Bispecific T-cell engagers non-covalently decorated drug-loaded PEGylated nanocarriers for cancer immunochemotherapy.

Immunotherapy is blooming in recent years. However, this therapy needs to overcome off-target effects, cytokine release syndrome, and low responses in the 'cold' tumor environment. Herein, various combinations of immunotherapies and chemotherapies were proposed to transform 'cold' tumors into 'hot' tumors to enhance the efficacy of immunotherapies. In this study, we prepared a biocompatible ganetespib (GSP)-loaded PEGylated nanocarriers (NCs) with a thin-film method, which exhibited a small particle size (~220.6 nm), high drug loading (~5.8%), and good stability. We designed and produced the cluster of differentiation 3 (CD3)/programmed death ligand 1 (PD-L1)/methoxy-polyethylene glycol (mPEG) trispecific antibodies (TsAbs) as bispecific T-cell engagers (BiTEs) to non-covalently bind the GSP-NCs via anti-mPEG fragment and endowed the GSP-NCs with a targeting ability and immunotherapeutic potential to activate cytotoxic T cells. Decoration of the GSP-NCs with TsAbs (BiTEs-GSP-NCs) significantly promoted the cellular uptake and showed synergistic effects through respective anti-PD-L1 and anti-CD3 activation of T cell-mediated cytotoxicity. In vivo tumor-inhibition studies also showed that the BiTEs-GSP-NCs could inhibit tumor growth with the GSP chemodrug and increase T-cell infiltration. This study provides a promising drug delivery strategy for cancer immunochemotherapy.

CPT11 with P-glycoprotein/CYP 3A4 dual-function inhibitor by self-nanoemulsifying nanoemulsion combined with gastroretentive technology to enhance the oral bioavailability and therapeutic efficacy against pancreatic adenocarcinomas.

Therapeutic efficacies of orally administrated hydrophobic chemodrugs are decreased by poor water solubilities and reduced oral bioavailabilities by P-glycoprotein (P-gp) and CYP450. In this study, CPT11 alone or combined with dual-function inhibitors (baicalein (BA) silymarin (SM), glycyrrhizic acid (GA), and glycyrrhetinic acid (GLA)) of P-gp and CYP450 loaded in a lecithin-based self-nanoemulsifying nanoemulsion preconcentrate (LBSNENP) to improve the solubility and inhibit the elimination by P-gp and CYP450. Results revealed that the LBSNENP composed of Capryol 90, lecithin/Tween 80/Cremophor EL, and propylene glycol at a weight ratio of 18:58:24 (designated PC90C10P0) was optimally selected. Encapsulating CPT11 with PEO-7000K in PC90C10P10/30 further enhanced the resultant hydrogel to be gastro-retainable and to release CPT11 in a sustained manner. Pharmacokinetic study of CPT11-loaded PC90C10P0 administered orally revealed an absolute bioavailability (FAB, vs. intravenous CPT11) of 7.8 ± 1.01% and a relative bioavailability (FRB1, vs. oral solution of CPT11) of 70.7 ± 8.6% with a longer half-life (T1/2) and mean residence time (MRT). Among the dual-function inhibitors, SM was shown to be the most influential in increasing the oral bioavailability of CPT11. SM also increased the plasma concentration of the SN-38 active metabolite, which formed from the enhanced plasma concentration of CPT11. It is concluded that treatment with CPT11 loaded in PC90C10P0 with or without solubilization with SM could expose tumors to higher plasma concentrations of both CPT11 and SN-38 leading to enhancement of tumor growth inhibition with no signs of adverse effects.

Active Tumoral/Tumor Environmental Dual-Targeting by Non-Covalently Arming with Trispecific Antibodies or Dual-Bispecific Antibodies on Docetaxel-Loaded mPEGylated Nanocarriers to Enhance Chemotherapeutic Efficacy and Minimize Systemic Toxicity.

PURPOSE: This study was aimed at developing the trispecific antibodies (anti-EGFR/anti-FAP/anti-mPEG, TsAb) or dual bispecific antibodies (anti-EGFR/anti-mPEG and anti-FAP/anti-mPEG) docetaxel (DTX)-loaded mPEGylated lecithin-stabilized micelles (mPEG-lsbPMs) for improving the targeting efficiency and therapeutic efficacy. METHODS: mPEG-lsbPMs were simply prepared via thin film method. The trispecific antibodies or bispecific antibodies bound the mPEG-lsbPMs by anti-mPEG Fab fragment. The formulations were characterized by DLS and TEM; in vitro and in vivo studies were also conducted to evaluate the cellular uptake, cell cytotoxicity and therapeutic efficacy. RESULTS: The particle sizes of mPEG-lsbPMs with or without the antibodies were around 100 nm; the formulations showed high encapsulation efficiencies of 97.12%. The TsAb and dual bispecific antibodies were fabricated and demonstrated their targeting ability. Two EGFR-overexpressed cell lines (HT-29 and MIA PaCa-2) were co-cultured with FAP-overexpressed WS1 cells (HT-29/WS1; MIA PaCa-2/WS1) to mimic a tumor coexisting in the tumor microenvironment. Cellular binding study revealed that the binding of anti-FAP micelles to three co-culture ratios (4:1, 1:1, and 1:4) of HT-29/EGFR to WS1/FAP was significantly higher than that for TsAb micelles and dual (1:1) micelles, and the binding of those targeting antibodies to WS1/FAP and MIA PaCa-2/EGFR was equally efficacious resulting in a similar binding amount of the TsAb and dual BsAbs (1:1) with the co-culture of MIA PaCa-2/EGFR and WS1/FAP at a 1:1 ratio. Antitumor efficacy study showed that treatment with DTX-loaded mPEG-lsbPMs modified with or without BsAbs, dual BsAbs (1:1), and TsAbs was enhanced in inhibiting tumor growth compared with that for Tynen® while showing fewer signs of adverse effects. CONCLUSION: Active targeting of both tumors and TAF-specific antigens was able to increase the affinity of DTX-loaded mPEG-lsbPMs toward tumor cells and TAFs leading to successive uptake by tumor cells or TAFs which enhanced their chemotherapeutic efficacy against antigen-positive cancer cells.

Preparation and Characterization of a Novel Swellable and Floating Gastroretentive Drug Delivery System (sfGRDDS) for Enhanced Oral Bioavailability of Nilotinib.

Regarding compliance and minimization of side effects of nilotinib therapy, there is a medical need to have a gastroretentive drug delivery system (GRDDS) to enhance the oral bioavailability that is able to administer an optimal dose in a quaque die (QD) or daily manner. In this study, the influence on a swelling and floating (sf) GRDDS composed of a polymeric excipient (HPMC 90SH 100K, HEC 250HHX, or PEO 7000K) and Kollidon® SR was examined. Results demonstrated that PEO 7000K/Kollidon SR (P/K) at a 7/3 ratio was determined to be a basic GRDDS formulation with optimal swelling and floating abilities. MCC PH102 or HPCsssl,SFP was further added at a 50% content to this basic formulation to increase the tablet hardness and release all of the drug within 24 h. Also, the caplet form and capsule form containing the same formulation demonstrated higher hardness for the former and enhanced floating ability for the latter. A pharmacokinetic study on rabbits with pH values in stomach and intestine similar to human confirmed that the enhanced oral bioavailability ranged from 2.65-8.39-fold with respect to Tasigna, a commercially available form of nilotinib. In conclusion, the multiple of enhancement of the oral bioavailability of nilotinib with sfGRDDS could offer a pharmacokinetic profile with therapeutic effectiveness for the QD administration of a reasonable dose of nilotinib, thereby increasing compliance and minimizing side effects.

Stearyl polyethylenimine complexed with plasmids as the core of human serum albumin nanoparticles noncovalently bound to CRISPR/Cas9 plasmids or siRNA for disrupting or silencing PD-L1 expression for immunotherapy.

PURPOSE: In this study, a double emulsion method for complexing plasmids with stearyl poly-ethylenimine (stPEI) as the core to form human serum albumin (HSA) (plasmid/stPEI/HSA) nanoparticles (NPs) was developed for gene delivery by non-covalently binding onto plasmid/stPEI/HSA nanoparticles with CRISPR/Cas9 or siRNA, which disrupts or silences the expression of programmed cell death ligand-1 (PD-L1) for immunotherapy. MATERIALS AND METHODS: Chemically synthesized stearyl-polyethyenimine (stPEI)/plasmids/HSA nanoparticles were maded by double emulsion method. They were characterized by dynamic light scattering (DLS), transmission electron microscope and also evaluated by in vitro study on CT 26 cells. RESULTS: stPEI was synthesized by an N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC)-N-hydroxysuccinimide (NHS) reaction, and we found that the degree of substitution was ~1.0 when the ratio of PEI to stearic acid was 1:7 in the reaction. Then, two sgRNA sequences were selected and evaluated for their ability to knock out PD-L1 by decreasing its expression by about 20%. Based on the trend of particle size/zeta potential values as a function of ratio, F25P1 containing 25 µg of plasmid/stPEI/HSA NPs noncovalently bound to 1 µg plasmids via charge-charge interactions was found to be optimal. Its particle size was about 202.7±4.5 nm, and zeta potential was 12.60±0.15 mV. In an in vitro study, these NPs showed little cytotoxicity but high cellular uptake. Moreover, they revealed the potential for transfection and PD-L1 knockout in an in vitro cell model. Furthermore, F25P1S0.5 containing 25 µg of plasmid/stPEI/HSA NPs noncovalently bound to 1 µg of plasmids and 0.5 µg siRNA was prepared to simultaneously deliver plasmids and siRNA. An in vitro study demonstrated that the siRNA did not interfere with the transfection of plasmids and showed a high-transfection efficiency with a synergistic effect on inhibition of PD-L1 expression by 21.95%. CONCLUSION: The plasmids/stPEI/HSA NPs could be a promising tool for gene delivery and improved immunotherapy.

[Genetic polymorphism of HSP70-1 gene and its correlation with resistance to mastitis in Chinese Holstein].

The polymorphisms of HSP70-1 gene in 253 Chinese Holstein dairy cows were studied, and the association between the polymorphisms and somatic cell score (SCS) were analyzed. PCR-SSCP, PCR-RFLP and DNA sequencing were used to investigate mutations in the coding region of HSP70-1 gene. The G-->A-->C mutation at 1 623 bp and G-->A mutation at 2 409 bp were found and both of them were silence mutations that caused no alteration in amino acid sequence. Chi-square test showed both loci were'nt at Hardy-Weinberg disequilibrium in Chinese Holstein. In the meanwhile, the association of 2 409 locus and SCS was not significant. However, the polymorphism at 1623 locus affected SCS significantly (P<0.05). The SCS of genotype CC was significantly lower than that of genotype AG and GG (P<0.05), so genotype CC was mastitis resistant. These results suggest that genotype CC of HSP70-1 gene may be used as a molecular and genetic marker to improve the phenotype of anti-mastitis in Chinese Holstein dairy cows.