Preparation of pH-sensitive nanogels bioconjugated with shark antibodies (VNAR) for targeted drug delivery with potential applications in colon cancer therapies.

Cancer is the second leading cause of death worldwide. To combat this disease, novel and specialized therapeutic systems are urgently needed. This is the first study to explore a system that combines shark variable domain (Fv) of new antigen receptor (VNAR) antibodies (hereinafter VNARs), PEGylated nanogels (pH-sensitive poly(N,N-diethylaminoethyl methacrylate, PDEAEM), and the anticancer drug 5-fluorouracil (5-FU) to explore its potential applications in colon cancer therapies. Nanogels were functionalized in a scalable reaction with an N-hydroxysuccinimide (NHS)-terminated polyethylene glycol derivative and bioconjugated with shark antibodies. Dynamic light scattering measurements indicated the presence of monodispersed nanogels (74 to 236 nm). All systems maintained the pH-sensitive capacity to increase in size as pH decreased. This has direct implications for the release kinetics of 5-FU, which was released faster at pH 5 than at pH 7.4. After bioconjugation, the ELISA results indicated VNAR presence and carcinoembryonic antigen (CEA) recognition. In vitro evaluations of HCT-116 colon cancer cells indicated that functionalized empty nanogels are not cytotoxic and when loaded with 5-FU, the cytotoxic effect of the drug is preserved. A 15% reduction in cell viability was observed after two hours of contact with bioconjugated nanogels when compared to what was observed with non-bioconjugated nanogels. The prepared nanogel system shows potential as an effective and site-specific nanocarrier with promising applications in in vivo studies of colon cancer therapies

Ca-Alginate-PEGMA Hydrogels for In Situ Delivery of TGF-β Neutralizing Antibodies in a Mouse Model of Wound Healing

Hydrogels provide effective alternatives for drug delivery when therapeutics cannot be applied directly to a wound, or if adverse effects are associated with systemic administration. However, drug delivery vehicles need to be biocompatible and biodegradable and exhibit sufficient mechanical strength to withstand handling and different physiological conditions, such as those encountered during topical administration of a therapeutic. Wound healing can be divided into three phases stimulated by transforming growth factor-beta (TGF-β) and, subsequently, targeted therapeutics have been developed to inhibit this cytokine for the treatment of chronic wounds and to prevent scarring. In this study, the capacity of calcium alginate hydrogels plasticized with poly(ethylene glycol) methyl ether methacrylate (PEGMA) to deliver anti-TGF-β antibodies (1D11.16.8) to a wound was investigated in situ. Three levels of antibodies, 10, 50, and 100 μg, were loaded into calcium-alginate-PEGMA hydrogels and evaluated in an excisional wound model in mice. Hydrogels containing 50 and 100 μg 1D11.16.8 produced less inflammation, accompanied by a marked reduction in collagen deposition and cell infiltration. These findings demonstrate the capacity of calcium-alginate-PEGMA hydrogels to deliver larger proteins, such as antibodies, to the site of a wound

Sizes of the PDEAEM:PEGMA:PEGA-NHS nanogels determined by dynamic light scattering (DLS).

a) Synthesis reproducibility and b) pH-responsive behavior of the nanogels.</p

<i>In vitro</i> analysis of VNAR-bioconjugated nanogels (N3).

A) ELISA of VNAR expression. On the horizontal axis: B (blank, Svelty milk 8%-PBS), N3 C- (nanogel without VNAR), C+ (CV043, 175 μg/mL), 1h (sample taken at hour 1 of the coupling reaction), 4h (sample taken at hour 4 of the coupling reaction), 8h (final sample taken at hour 8 of the coupling reaction), and PF (final product after dialysis [N3+VNAR]). B) ELISA assay of CEA recognition. On the horizontal axis: VNAR (CV043, 175 μg/mL), B (blank, Svelty milk 8%-PBS), c) cell viability by aqueous one solution cell proliferation assay (MTS) of empty selected nanogels at 24h, PBS (C-), DMSO 5% (C+), d) cell viability by MTS of 5-FU and nanogels containing 5-FU. On the horizontal axis: B (PBS 7.4), N3 (empty nanogels, 5-FU [20 μg/mL]), N3-VNAR-5FU (20 μg 5FU/mL), and N3-5-FU (20 μg 5FU/mL). ANOVA test, *p < 0.01, **p < 0.001, and ***p < 0.0001 versus N3 and PBS (C-). PDEAEM: poly(N,N-diethylaminoethyl methacrylate), PEGMA: poly(ethylene glycol) methyl ether methacrylate, PEGA-NHS: Acrylate-PEG3500-NHS, and CEA: carcinoembryonic antigen.</p

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Cancer is the second leading cause of death worldwide. To combat this disease, novel and specialized therapeutic systems are urgently needed. This is the first study to explore a system that combines shark variable domain (Fv) of new antigen receptor (VNAR) antibodies (hereinafter VNARs), PEGylated nanogels (pH-sensitive poly(N,N-diethylaminoethyl methacrylate, PDEAEM), and the anticancer drug 5-fluorouracil (5-FU) to explore its potential applications in colon cancer therapies. Nanogels were functionalized in a scalable reaction with an N-hydroxysuccinimide (NHS)-terminated polyethylene glycol derivative and bioconjugated with shark antibodies. Dynamic light scattering measurements indicated the presence of monodispersed nanogels (74 to 236 nm). All systems maintained the pH-sensitive capacity to increase in size as pH decreased. This has direct implications for the release kinetics of 5-FU, which was released faster at pH 5 than at pH 7.4. After bioconjugation, the ELISA results indicated VNAR presence and carcinoembryonic antigen (CEA) recognition. In vitro evaluations of HCT-116 colon cancer cells indicated that functionalized empty nanogels are not cytotoxic and when loaded with 5-FU, the cytotoxic effect of the drug is preserved. A 15% reduction in cell viability was observed after two hours of contact with bioconjugated nanogels when compared to what was observed with non-bioconjugated nanogels. The prepared nanogel system shows potential as an effective and site-specific nanocarrier with promising applications in in vivo studies of colon cancer therapies.</div

Model structures for bioconjugation of PDEAEM:PEGMA:PEGA-NHS nanogels.

A) UV-Vis of folic acid-bioconjugated nanogels. B) Fluorescence emission spectra of GFP-bioconjugated nanogels.</p

General characteristics of PDEAEM-based nanogels.

Cancer is the second leading cause of death worldwide. To combat this disease, novel and specialized therapeutic systems are urgently needed. This is the first study to explore a system that combines shark variable domain (Fv) of new antigen receptor (VNAR) antibodies (hereinafter VNARs), PEGylated nanogels (pH-sensitive poly(N,N-diethylaminoethyl methacrylate, PDEAEM), and the anticancer drug 5-fluorouracil (5-FU) to explore its potential applications in colon cancer therapies. Nanogels were functionalized in a scalable reaction with an N-hydroxysuccinimide (NHS)-terminated polyethylene glycol derivative and bioconjugated with shark antibodies. Dynamic light scattering measurements indicated the presence of monodispersed nanogels (74 to 236 nm). All systems maintained the pH-sensitive capacity to increase in size as pH decreased. This has direct implications for the release kinetics of 5-FU, which was released faster at pH 5 than at pH 7.4. After bioconjugation, the ELISA results indicated VNAR presence and carcinoembryonic antigen (CEA) recognition. In vitro evaluations of HCT-116 colon cancer cells indicated that functionalized empty nanogels are not cytotoxic and when loaded with 5-FU, the cytotoxic effect of the drug is preserved. A 15% reduction in cell viability was observed after two hours of contact with bioconjugated nanogels when compared to what was observed with non-bioconjugated nanogels. The prepared nanogel system shows potential as an effective and site-specific nanocarrier with promising applications in in vivo studies of colon cancer therapies.</div

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Nanogels synthesized by surfactant-free emulsion polymerization (SFEP): a) Chemical structures for PDEAEM:PEGMA:PEGA-NHS nanogels, APS is (NH4+)2(S2O8)2-, b) Nanogels bioconjugated with shark antibodies: PDEAEM:PEGMA:PEGA-VNAR-5FU. Poly(N,N-diethylaminoethyl methacrylate) (PDEAEM, black), poly(ethylene glycol) methacrylate (PEGMA, red), acrylate-PEG3500-NHS (PEGA-NHS, blue), variable domain of new antigen receptor (VNAR, anchor), ethyleneglycol dimethacrylate (EGDMA, green), and 5-fluorouracil (5-FU, anticancer drug, yellow circle).</p

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Cancer is the second leading cause of death worldwide. To combat this disease, novel and specialized therapeutic systems are urgently needed. This is the first study to explore a system that combines shark variable domain (Fv) of new antigen receptor (VNAR) antibodies (hereinafter VNARs), PEGylated nanogels (pH-sensitive poly(N,N-diethylaminoethyl methacrylate, PDEAEM), and the anticancer drug 5-fluorouracil (5-FU) to explore its potential applications in colon cancer therapies. Nanogels were functionalized in a scalable reaction with an N-hydroxysuccinimide (NHS)-terminated polyethylene glycol derivative and bioconjugated with shark antibodies. Dynamic light scattering measurements indicated the presence of monodispersed nanogels (74 to 236 nm). All systems maintained the pH-sensitive capacity to increase in size as pH decreased. This has direct implications for the release kinetics of 5-FU, which was released faster at pH 5 than at pH 7.4. After bioconjugation, the ELISA results indicated VNAR presence and carcinoembryonic antigen (CEA) recognition. In vitro evaluations of HCT-116 colon cancer cells indicated that functionalized empty nanogels are not cytotoxic and when loaded with 5-FU, the cytotoxic effect of the drug is preserved. A 15% reduction in cell viability was observed after two hours of contact with bioconjugated nanogels when compared to what was observed with non-bioconjugated nanogels. The prepared nanogel system shows potential as an effective and site-specific nanocarrier with promising applications in in vivo studies of colon cancer therapies.</div