Peptide, polyamidoamine dendrimers and protein functionalized polycaprolactone nanoparticles and their mechanism of cellular uptake

Abstract: Polycaprolactone (PCL) as degradable aliphatic polyester and its block copolymers are currently being widely explored in the formulation of nanoparticles (NPs) for targeted therapies, due to its biodegradability, biodegradation kinetics, compatibility with drugs, and an acceptable safety profile. The uptake of NPs is mediated by cell-membrane interactions and their intracellular transport and fate is determined by the transporters it engages on the cell plasma membrane. However, PCL lacks chemistry for functionalization and easy introduction of functional groups targeting receptors onto NPs could significantly increase their utility in targeted drug delivery. In this regard, past work from our laboratory has shown polycaprolactone copolymer bearing alkyne onto a hydrophilic spacer in the backbone could be utilized in either a post modification in organic solvent or preparing NPs, which could be direct modified in aqueous environment via click reaction. The focus of this thesis is to optimize the functionalization of NPs with cell interaction peptide and proteins and better understand how NPs with the surface-bound information interacted with cell membrane and how NPs enter and get trafficked in cells.
In the first part of this thesis, the synthesis of PCL copolymer presenting alkyne group at the terminal end of hydrophilic spacer is described and the post-functionalization of NPs produced using these alkyne-bearing PCL with a cRGD (cyclic-arginine-glycine-aspartic acid) is described and characterized; and the uptake of NPs in several cell systems is presented. NPs in the size range of 146.4±4.4 nm and very narrow polydispersity index of 0.150±0.02 were successfully prepared by the nanoprecipitation. These NPs surfaces showed an alkyne rich surface. The surface density or concentration of alkyne groups was determined using covalent reaction between azide-Cy3-sulfo and was found to be greater than 52 nmol of alkyne group in 500 μl NPs suspension (307.8 μg PCL copolymer inside), which corresponds to ~ 50.4% in total of alkyne group in the polymer on the NPs surface. RGD as a fundamental recognition site for the attachment of proliferating endothelial to the extracellular matrix is ideal as tumor-targeting ligands for NPs. The cyclic variant of RGD was introduced on to the NPs surface via Cu (I)-catalyzed click reaction (Huigsen’s 1,3-dipolar cycloaddition) using Cyclo [Arg-Gly-Asp-D-Phe-Lys(PEG-PEGAzide)]. The presence of cRGD on the NP surface was confirmed using infrared spectroscopy. In order to unveil the suitability of this modified polymer as nanocarriers and understand how cRGD influences NPs trafficking, their uptake behavior was studied in epithelial and endothelial cells systems. Human cervical cancer epithelial cells (HeLa), human pulmonary adenocarcinoma cells (A549), human triple-negative breast cancer cells (MDA-MB-231), human primary pulmonary microvascular endothelial cells (HPMEC) and human umbilical vein endothelial cell (HUVEC) were treated with NPs with and without cRGD modification. In general cRGD modified led to a decrease in uptake in epithelial cells, however a differential uptake was observed in HPMEC and HUVEC with presence of cRGD increasing the uptake in HUVEC. Western blot analysis implicates a role for integrin β1 that inhibits transdominate of integrin β3 in the observed phenomenon.
In the second part of this thesis, the synthesis of different generations of aizde-polyamidoamine (azido PAMAM) dendrimers (D0, D1, D2, D3) through alternating-sequential Michael addition between the amino-terminated and methyl acrylate, resulting in an ester-terminated outer layer and coupling with ethylene diamine to achieve a new amino-terminated surface is described. Aqueous suspensions of the aforementioned alkyne presenting PCL NPs were modified with azido PAMAMs via "click reaction". The amino groups on the dendrimer modified NPs (PAMAM_NPs) are presented at various distances determined by the dendrimer generation. Generation and can be exploited to study the role of receptor clustering on NP uptake. Cholera Toxin B subunit (CTB), which is known to internalize in cells via caveolae was introduced onto the NP surface through coupling via carboxylic acid groups on the CTB using EDC (1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride/NHS (N-hydroxysulfosuccini--mide (Sulfo-NHS)) chemistry. The uptake behavior of CTB modified PAMAM-NPs was compared to unmodified PAMAM_NPs in HeLa cells. The introduction of CTB was found to retard the uptake of NPs and a role for phosphorylation of caveolin-1 is proposed based in western blot analysis and fluorescence microscopy in cells.
In the third part of this thesis, synthesis and characterization of a polycaprolactone bearing PAMAM in the side chain of the backbone is described. A series of amine-bearing NPS were prepared by nanoprecipitation and the surface amine concentration was determined using sulfosuccinimidyl-4-o-(4,4-dimethoxytrityl) butyrate (sulfo-SDTB) assay. The cell viability and uptake of these NPs were undertaken in HeLa, A549 cells and MDA-MB-231

Angaben zum Objekt

• Standort: Deutsche Nationalbibliothek Frankfurt am Main
• Umfang: Online-Ressource
• Sprache: Englisch
• Anmerkungen: Universität Freiburg, Dissertation, 2019

Klassifikation und Themen

• Schlagwort: Nanoparticles ; Polycaprolactone ; Dendrimers ; Peptide ; RGD-Sequenz ; Polycaprolactone ; Nanopartikel ; Sternpolymere

Beteiligte, Orts- und Zeitangaben

• Ereignis: Veröffentlichung
• (wo): Freiburg
• (wer): Universität
• (wann): 2019
• Urheber: Zhang, Weihai
• Beteiligte Personen und Organisationen: Shastri, V. Prasad ; Albert-Ludwigs-Universität Freiburg. Fakultät für Chemie und Pharmazie

Weitere Informationen

• DOI: 10.6094/UNIFR/150691
• URN: urn:nbn:de:bsz:25-freidok-1506913
• Rechteinformation: Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Letzte Aktualisierung: 14.08.2025, 10:56 MESZ