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Physicochemical properties and protein interaction of self-assembled gelatin oleic nanoparticles

초록/요약

After administration nanoparticle (NP) into biological fluids, NP- protein complex forms and represents “true identity” of NP in our body. The protein corona alters the size and interface composition of a nanomaterial, giving it a new biological identity that is seen by cells. The biological identity determines the physiological response, including agglomeration, cellular uptake, circulation lifetime, kinetics, transport, accumulation, and toxicity. Hence, protein-NP interaction should be carefully investigated to predict and control the fate of NPs including distribution and bioavailability. A numerous studies evaluated the effect of protein on the cellular behavior but mainly using inorganic NPs. In this studied, self-assembled gelatin oleic acid nanoparticles, a polymeric system was used to initial characterization of the effect induce by treatment condition, including media and the presence of protein, mainly BSA. Amphiphilically-modified gelatin contains an inner core that providing a hydrophobic domain to accommodate water-insoluble drugs, whereas the outer gelatin layer accounting for the enhanced biocompatibility and faster biodegradability. Gelatin-oleic acid conjugates (GOC) was synthesized from oleic acid (OA) and gelatin, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) in 60% ethanol. The optimized process for the GOC synthesis was well established. Self-assembled gelatin-oleic acid nanoparticles (GON) were fabricated simply by the desolvation method using dialysis membrane, without adding a cross-linker. GON were formed at a lower critical micelle concentration (CMC) in water, 0.32 mg/mL and homogenous size of 100-300 nm. The GON showed no toxicity in human embryonic kidney cells (HEK 293) and significantly improved coumarin loading in adenocarcinomic human alveolar basal epithelial cells (A549). The current GOC and GON could provide versatile strategies to deliver cargo into the body by acting as a safe and biocompatible nanoparticulate carrier. When administrated, nanoparticles (NPs) will be affected by environment conditions such as pH, composition, temperature and especially proteins. Gelatin-oleic nanoparticles (GON) and bovine serum albumin (BSA), the highest abundance protein in blood were chosen to be evaluated in this study. Influence of BSA at various concentrations on GON stability was characterized by spectroscopy methods. The cellular associations of GON on HEK 293 and A549 cell lines were studied, in two common cell culture media DMEM and RPMI, with and without the addition of BSA at various concentrations. The presence of BSA reduced cellular uptake on both HEK 293 and A549 cell lines in both types of media. However, in the media supplementary with 10% FBS, there was no observation of reduction in cellular uptake, except for media with addition of 10 mg/mL BSA.Preformed corona GON by adsorption of BSA induced lower cellular uptake of GON in A549 cells in RPMI with and without FBS. Therefore, BSA can be used as endogenous ligand applied in NP design to reduce cellular uptake simply by mixing or incubation, but still required thoroughly assessment.

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목차

Table of content
Chapter 1
Synthetic optimization of gelatin-oleic conjugate and aqueous-based formation of self-assembled nanoparticles without cross-linkers
Abstract
1. Introduction
2. Materials & methods
2.1. Materials
2.2. Synthesis of GOC
2.2.1. Activation of oleic acid by EDC/NHS
2.2.2. Conjugation of gelatin with OA
2.3. Characterization of GOC
2.3.1. Fourier transform infrared spectroscopy (FT-IR)
2.3.2. 1H NMR Spectroscopy
2.3.3. Determination of the substitution degree of GOC
2.4. Preparation of self-assembled GON
2.5. Characterization of GON
2.5.1. Evaluation of the critical micelle concentration (CMC)
2.5.2. Physical characterization of the self-assembled nanoparticles
2.5.3. Fluorescence spectra of GON
2.5.4. Stability of GON
2.5.5. Cell cytotoxicity
2.5.6. Cellular uptake
2.6. Statistical study
3. Results and Discussion
3.1. GOC synthetic optimization.
3.1.1. Effect of the synthesis parameters on GOC substitution
3.1.2. Characterization of GOC
3.2. Characterizations of GON
3.2.1. Self-assembly behavior of GOC
3.2.2. Fluorescence spectrum of GON
3.2.3. Physical characteristics of GON
3.2.4. Stability test
3.2.5. Cytotoxicity test
3.2.6. Cellular uptake
4. Conclusions
References
Chapter 2
Effect of bovine serum albumin on the physicochemical properties and cellular association of self-assembled gelatin-oleic nanoparticles
Abstract
1. Introduction
2. Materials & methods
2.1. Materials
2.2. Preparation of GONs and coumarin 6-loaded GONs
2.2.1. Gelatin-oleic conjugate (GOC) synthesis
2.2.2. GON preparation
2.2.3. Preparation of coumarin 6-loaded GONs
2.2.4. Preparation of BSA pre-coated GONs
2.3. Physicochemical characterization of the GONs
2.3.1. Size and zeta potential characterization
2.3.2. Transmission electron microscopy (TEM)
2.3.3. Fluorescence spectra BSA-GON complexes
2.4. Cellular uptake of GONs and GON-BSA complexes
2.4.1. Cell culture
2.4.2. Treatment of cells with GONs and BSA pre-coated GONs
2.4.3. Flow cytometry
2.4.4. Confocal microscopy
2.5. Statistical analysis
3. Results and Discussion
3.1. GON fabrication
3.2. Interaction between BSA and GONs
3.2.1. Fluorescence spectra of BSA-GON complexes
3.2.2. Effect of BSA on the physicochemical properties of GONs
3.3. Effect of BSA on the cellular uptake of GONs
3.3.1. Effect of the presence of BSA in the growth media on GON uptake
3.2.2. Effect of BSA pre-coating on GON cellular uptake
4. Conclusions
References

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