Temperature-Responsive Magnetic Nanoparticles for Bioanalysis of Lysozyme in Urine Samples

Highly selective multifunctional magnetic nanoparticles containing a thermoresponsive polymer shell were developed and used in the sample pretreatment of urine for the assessment of lysozymuria in leukemia patients. Crosslinked poly(N-isopropylacrylamide-co-acrylic acid-co-N-tert-butylacrylamide) was grown onto silica-coated magnetic nanoparticles by reversible addition fragmentation chain transfer (RAFT) polymerization. The lysozyme binding property of the nanoparticles was investigated as a function of time, protein concentration, pH, ionic strength and temperature and their selectivity was assessed against other proteins. High-abundant proteins, like human serum albumin and γ-globulins did not interfere with the binding of lysozyme even at elevated concentrations characteristic of proteinuria. A sample cleanup procedure for urine samples has been developed utilizing the thermocontrollable protein binding ability of the nanoparticles. Method validation was carried out according to current bioanalytical method validation guidelines. The method was highly selective, and the calibration was linear in the 25 to 1000 µg/mL concentration range, relevant in the diagnosis of monocytic and myelomonocytic leukemia. Intra- and inter-day precision values ranged from 2.24 to 8.20% and 1.08 to 5.04%, respectively. Intra-day accuracies were between 89.9 and 117.6%, while inter-day accuracies were in the 88.8 to 111.0% range. The average recovery was 94.1 ± 8.1%. Analysis of unknown urine samples in comparison with a well-established reference method revealed very good correlation between the results, indicating that the new nanoparticle-based method has high potential in the diagnosis of lysozymuria.

Thermocontrolled Reversible Enzyme Complexation-Inactivation-Protection by Poly(N-acryloyl glycinamide)

A prospective technology for reversible enzyme complexation accompanied with its inactivation and protection followed by reactivation after a fast thermocontrolled release has been demonstrated. A thermoresponsive polymer with upper critical solution temperature, poly(N-acryloyl glycinamide) (PNAGA), which is soluble in water at elevated temperatures but phase separates at low temperatures, has been shown to bind lysozyme, chosen as a model enzyme, at a low temperature (10 °C and lower) but not at room temperature (around 25 °C). The cooling of the mixture of PNAGA and lysozyme solutions from room temperature resulted in the capturing of the protein and the formation of stable complexes; heating it back up was accompanied by dissolving the complexes and the release of the bound lysozyme. Captured by the polymer, lysozyme was inactive, but a temperature-mediated release from the complexes was accompanied by its reactivation. Complexation also partially protected lysozyme from proteolytic degradation by proteinase K, which is useful for biotechnological applications. The obtained results are relevant for important medicinal tasks associated with drug delivery such as the delivery and controlled release of enzyme-based drugs.

Instantaneous Degelling Thermoresponsive Hydrogel

Responsive polymeric hydrogels have found wide application in the clinic as injectable, biocompatible, and biodegradable materials capable of controlled release of therapeutics. In this article, we introduce a thermoresponsive polymer hydrogel bearing covalent disulfide bonds. The cold aqueous polymer solution forms a hydrogel upon heating to physiological temperatures and undergoes slow degradation by hydrolytic cleavage of ester bonds. The disulfide functionality allows for immediate reductive cleavage of the redox-sensitive bond embedded within the polymer structure, affording the option of instantaneous hydrogel collapse. Poly (ethylene glycol)-b-poly (lactic acid)-S-S-poly (lactic acid)-b-poly (ethylene glycol) (PEG-PLA-SS-PLA-PEG) copolymer was synthesized by grafting PEG to PLA-SS-PLA via urethane linkages. The aqueous solution of the resultant copolymer was a free-flowing solution at ambient temperatures and formed a hydrogel above 32 °C. The immediate collapsibility of the hydrogel was displayed via reaction with NaBH4 as a relatively strong reducing agent, yet stability was displayed even in glutathione solution, in which the polymer degraded slowly by hydrolytic degradation. The polymeric hydrogel is capable of either long-term or immediate degradation and thus represents an attractive candidate as a biocompatible material for the controlled release of drugs.

Enhanced Delivery of Thermoresponsive Polymer-Based Medicine into Tumors by Using Heat Produced from Gold Nanorods Irradiated with Near-Infrared Light

The aim of this study was to establish a drug delivery system (DDS) for marked therapy of tumors using a thermoresponsive polymer, polyoxazoline (POZ). The effectiveness of the following was investigated: (i) the delivery of gold nanorods (GNRs) to tumor tissues, (ii) heat production of GNR upon irradiation with near-infrared (NIR) light, and (iii) high accumulation of an intravenously injected radiolabeled POZ as a drug carrier in tumors by sensing heat produced by GNRs. When the GNR solution was irradiated with NIR light (808 nm), the solution temperature was increased both in a GNR-concentration-dependent manner and in a light-dose-dependent manner. POZ, with a lower critical solution temperature of 38 °C, was aggregated depending on the heat produced by the GNR irradiated by NIR light. When it was intratumorally pre-injected into colon26-tumor-bearing mice, followed by NIR light irradiation (GNR+/Light+ group), the tumor surface temperature increased to approximately 42 °C within 5 min. Fifteen minutes after irradiation with NIR light, indium-111 (111In)-labeled POZ was intravenously injected into tumor-bearing mice, and the radioactivity distribution was evaluated. The accumulation of POZ in the tumor was significantly (approximately 4-fold) higher than that in the control groups (GNR+/without NIR light irradiation (Light–), without injection of GNR (GNR–)/Light+, and GNR–/Light– groups). Furthermore, an in vivo confocal fluorescence microscopy study, using fluorescence-labeled POZ, revealed that uptake of POZ by the tumor could be attributed to the heat produced by GNR. In conclusion, we successfully established a novel DDS in which POZ could be efficiently delivered into tumors by using the heat produced by GNR irradiated with NIR light.

Spectroscopic Fourier Transform Infrared Study of Poly (N-Isopropylacrylamide)-Human Recombinant Platelet-Derived Growth Factor- BB Protein as a Strategic Biobetter Regenerative Material

Biobetter or Biosuperior are recombinant protein drugs with one or more characteristics that are better than the original formulation. Platelet-Derived Growth Factor (PDGF) is a potent mitogen that can induce pulp stem cell proliferation and promote angiogenesis in vitro. PDGF-BB is required for wound healing, and they are chemoattractants that can activate macrophages and fibroblast activation in vitro. Poly(n-isopropylacrylamide) (PNIPAM) polymer is a thermoresponsive polymer widely used for drug delivery. A lyophilized combination of PNIPAM and 5µg of Recombinant Human PDGF-BB(rhPDGF-BB)(sigma Aldrich-P3201) protein was prepared in the present study. This combination was subjected to Spectroscopic Fourier-Transform Infrared(FTIR) study. The results suggested that a biophysical approach like FTIR enables assessing protein stability and aggregation tendency of recombinant proteins, contributing as a useful method to identify samples with prospective high therapeutic values.