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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

<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

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

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

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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

Analysis of functionalized nanogels (N3), VNAR-bioconjugated nanogels, and nanogels loaded with 5-FU.

A) Size distribution by dynamic light scattering (DLS) measurements at pH 8, b) UV-Vis absorbance at 280 nm, c) cumulative 5-FU release at pH 7.4 and pH 5, and d) nanogel dispersion (N3+5FU, pH 7.4) and model of chemical interactions between PDEAEM-based nanogels and 5-FU.</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