PASSIVATION AND FUNCTIONALIZATION OF CONJUGATED POLYMER NANOPARTICLES WITH HEAD GROUP MODIFIED PHOSPHOLIPIDS AND PROTEINS

Conjugated polymer nanoparticles (CPNs) possess important characteristics such as high fluorescence brightness, reasonable photostability, and non-toxicity. These properties allow the use of CPNs in fluorescence based cellular and biological applications including cellular labeling, imaging, biosensing, and single particle tracking. To realize the broad applications of CPNs, it is required that CPNs possess functionality to conjugate a recognition moiety and maintain colloidal stability in biological media. In the following Dissertation, we have prepared functionalized CPNs by surface passivation with head group- functionalized poly (ethylene glycol) lipids and proteins. We studied the colloidal stability of CPNs in biological media and investigated their utility as cellular labels, fluid phase markers and detection reagent in immunoassay. Chapter 1 summarizes the general background information of CPNs including methods of preparation, physical properties, bioanalytical applications, and functionalization strategies. Chapter 2 contains a systematic study of a simple and rapid method to prepare extremely bright, functionalized, stable and biocompatible conjugated polymer nanoparticles incorporating functionalized polyethylene glycol (PEG) lipids by reprecipitation. The size of these nanoparticles, as determined by TEM, was 24±5 nm. These nanoparticles retain the fundamental spectroscopic properties of conjugated polymer nanoparticles prepared without PEG lipids, but demonstrate greater hydrophilicity and quantum yield compared to unmodified conjugated polymer nanoparticles. Nanoparticles were prepared with several PEG lipid functional end groups, including biotin and carboxy moieties that can then be conjugated to biomolecules. We have demonstrated the availability of these end groups for functionalization using the interaction of biotin PEG lipid conjugated polymer nanoparticles with streptavidin. To demonstrate that nanoparticle functionalization could be used for targeted labeling of specific cellular proteins, biotinylated PEG lipid conjugated polymer nanoparticles were bound to biotinylated anti-CD 16/32 antibodies on J774A.1 cell surface receptors, using streptavidin as a linker. This work represents a method of preparation of bright and biocompatible CPNs by inclusion of functionalized PEG lipids. The functional end group on PEG lipid CPNs offers a link to conjugate CPNs to biomolecules. Hence, PEG-lipid CPNs are a viable technology for targeted labeling and imaging in biological systems. Chapter 3 constitutes the comparative study of sensitivity and limit of detection using PEG-lipid functionalized CPNs as a fluid phase marker in J774A.1 cells compared to cells loaded with carboxy-functionalized quantum dots (Q dots) or a dextran-linked small molecule organic dyes (Alexa fluor 488 dextran (AF488-dex)). Under typical conditions used for ex vivo biological imaging or flow cytometry, these CPNs were 175x brighter than Qdots and 1,400x brighter than AF488-dex. Evaluation of the minimum incubation concentration required for detection of nanoparticle fluorescence with a commercial flow cytometer indicated that the limit of detection for PEG lipid-PFBT CPNs was 19 pM( 86 ppb), substantially lower than values obtained for Q dots (980 pM) or AF488-dex (11.2 nM). Taken together, these data clearly indicate that CPNs can be used at very low labeling concentrations and are excellent fluid- phase markers with significantly greater fluorescence brightness than existing dyes or nanoparticles. Chapter 4 studies the protein passivation of CPNs as a reliable approach to provide the colloidal stability of conjugated polymer nanoparticles in tissue culture media and buffer solutions. Unmodified CPNs aggregate under physiological salt conditions and are therefore unsuitable for biological applications such as imaging and sensing. We showed that when incubated in protein solutions (bovine albumin serum, lysozyme, or fetal bovine serum), bare CPNs rapidly acquire a stable protein coat that both increases CPN diameter and prevents aggregation at physiological ionic strength over pH range of 4 - 8. The protein coat is highly stable; no change in hydrodynamic diameter is observed upon extended incubation following size exclusion chromatography into protein free saline solution. The results show that adsorption of protein on CPN surface does not alter fluorescence brightness. BSA-biotin modified CPNs show availability of protein corona for molecular recognition. Hence, we concluded that protein adsorption is a simple method to provide colloidal stability in physiological buffer and to modify CPNs for target selective cellular imaging and sensing. Finally, Chapter 5 reports the study of protein coating as a general method for providing functionality in CPNs. We have demonstrated that protein-A coated CPNs serve as scaffold for CPN linkage to IgG. Using anti-rabbit IgG linked CPNs as a detection reagent; we have detected rabbit IgG in solid phase immunoassay with antigen-antibody binding constant of 5.5 ± 0.8 nM. Similarly, neutravidin coated CPNs that conjugated to biotin linked recognition moiety also serve as a direct detection reagent. Together, we conclude that protein A, neutravidin and immunoglobulin modified CPNs serve as direct detection reagent in solid phase based immunoassays. Taken together, this study shows that head group functionalized phospholipids and a broad variety of proteins readily modify the surface of hydrophobic conjugated polymer nanoparticles. The resulting CPNs retain and improve fluorescence brightness. Hence, head group functionalized phospholipids and proteins act as linkers for biomolecules on the nanoparticle surface. Such CPNs are bright photon source for specific labeling, imaging, and sensing applications