Regulating protein corona on nanovesicles by glycosylated polyhydroxy polymer modification for efficient drug delivery

Nanocarriers, upon entering the human body, inevitably adsorb numerous proteins from bodily fluids, forming a dynamic protein corona. This corona alters the nanocarrier's biological identity, influencing its behavior and presenting a significant challenge to effective drug delivery. The corona's dynamic nature, constantly exchanging proteins as the nanocarrier travels through various physiological environments, leads to immune system recognition and clearance, hindering targeted delivery to desired cells. While factors affecting corona formation have been studied, effectively modulating the interaction of functional proteins on the nanosurface remains a challenge. Polyhydroxy polymers like PEG are frequently used to modify nanocarriers to reduce protein adsorption, but PEGylation can trigger the production of anti-PEG antibodies, leading to accelerated blood clearance (ABC) upon repeated administration. This necessitates exploring alternative surface modifications. Previous research suggests that the interaction between nanocarriers and proteins influences cellular internalization. Manipulating the protein corona could enhance cellular uptake in tumor cells. For instance, functionalizing nanoparticles with penicillamine to adsorb transferrin enhances interaction with the overexpressed transferrin receptor on cancer cells. Also, cationic polymers can facilitate passive uptake in tumor cells. Hydroxyl and amino groups, key components of hydrogen bonds in protein-receptor interactions, offer an alternative approach to modifying polymers and regulating nanocarrier-protein interactions. This study aims to investigate the influence of glycosylated polyhydroxy polymers (CSO-g-TCP) with varying amino/hydroxyl ratios on the protein corona of lipid nanovesicles (CP-LVs). CP-LVs with an optimal amino/hydroxyl ratio are expected to reduce protein corona formation in plasma and liver, preventing immune activation and ABC. Simultaneously, they are expected to adsorb tumor-associated proteins, improving selective uptake in tumor cells and resulting in enhanced antitumor efficacy when loaded with paclitaxel.

Extensive research demonstrates the significant impact of the protein corona on the in vivo behavior of nanocarriers. The dynamic exchange of proteins within the corona as nanocarriers traverse different biological environments is a key factor influencing their fate. Studies have shown that the protein corona can lead to immune system recognition and clearance, reducing the efficacy of drug delivery systems. While PEGylation has been widely employed to mitigate protein adsorption and prolong circulation time, it suffers from limitations such as the induction of anti-PEG antibodies and subsequent accelerated blood clearance (ABC) following repeated administrations. This necessitates the exploration of alternative strategies for surface modification to overcome the limitations of PEGylation. The literature suggests that controlling the protein corona composition might improve targeted drug delivery. For instance, specific surface modifications can selectively adsorb target-specific proteins which increases cellular internalization in tumor cells. Different chemical modifications can influence the protein adsorption pattern and subsequent in vivo biological behavior. Therefore, controlling the protein corona through suitable surface modification could be a promising strategy to improve the efficiency of drug delivery.

Glycosylated polyhydroxy polymers (CSO-g-TCP) were synthesized by grafting chitosan oligosaccharide (CSO) onto PEO-PPO-PEO triblock copolymers (TCP) to create lipid nanovesicles (CP-LVs) with different amino/hydroxyl ratios. The amino/hydroxyl ratios were determined by Boehm titration and confirmed by time-of-flight secondary ion mass spectrometry (ToF-SIMS) which showed that the hydroxyl groups were distributed throughout the modified layers, while amino groups were primarily on the outer leaflet of the membrane. The size, zeta potential, and morphology of the CP-LVs were characterized. The composition of the protein corona formed on CP-LVs after incubation with plasma and liver tissue interstitial fluid (TIF) was analyzed using the bicinchoninic acid (BCA) assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and Western blotting. The dynamic changes in protein corona composition during the transition from plasma to liver were investigated using a plasma-PBS-liver TIF incubation system. Isothermal titration calorimetry (ITC) and confocal laser scanning microscopy (CLSM) were used to measure the binding affinities and visualize the interactions between proteins (albumin and IgG) and nanovesicles. In vivo studies in mice were performed to analyze the protein corona composition in plasma and liver after single and repeated administrations. Macrophage uptake of nanovesicles was evaluated using J774 cells (blood macrophages) and Kupffer cells (liver macrophages). Pharmacokinetics and biodistribution studies were conducted to determine the blood circulation time and tissue distribution of the nanovesicles. The effect of the protein corona on tumor cell internalization was investigated by incubating nanovesicles with tumor TIF and evaluating cellular uptake in A549 and HeLa cells. The roles of OPN and CD44 were investigated using inhibitors (cilengitide and hyaluronic acid). The antitumor efficacy of paclitaxel-loaded CP₁-LVs was evaluated in A549 and HeLa tumor-bearing mice, assessing tumor growth, survival, and organ histology.

CP₁-LVs, with an amino/hydroxyl ratio of approximately 0.4, exhibited significantly reduced protein adsorption in plasma and liver compared to control groups (PEG-Lips and Lips). CP₁-LVs showed the lowest adsorption of immunoglobulins (IgG and IgM) in both plasma and liver TIF, leading to prolonged blood circulation and reduced macrophage uptake. In contrast to PEG-Lips, which displayed accelerated blood clearance (ABC) upon repeated administration, CP₁-LVs maintained prolonged blood circulation. When transported to tumors, CP₁-LVs selectively adsorbed abundant tumor-specific proteins, such as CD44 and osteopontin (OPN). This selective adsorption enhanced tumor cell internalization. The differential protein adsorption (low IgG, high CD44/OPN) on CP₁-LVs in tumor versus liver environments was attributed to the varying isoelectric points (pI) of these proteins and their electrostatic interactions with the nanovesicle surface. In vitro studies showed that paclitaxel-loaded CP₁-LVs displayed significantly enhanced cytotoxicity against A549 and HeLa cells compared to paclitaxel-loaded PEGylated liposomes. In vivo studies in tumor-bearing mice demonstrated that CP₁-LVs accumulated more effectively in tumors than PEGylated liposomes, resulting in significantly enhanced antitumor efficacy with prolonged survival time and no apparent organ toxicity.

This study demonstrates that precisely controlling the composition of the protein corona on nanocarriers is crucial for optimizing drug delivery. The findings highlight the limitations of PEGylation, specifically the induction of anti-PEG antibodies and the subsequent ABC effect. The glycosylated polyhydroxy polymer modification strategy, particularly with CP₁-LVs, offers a superior alternative by minimizing protein adsorption, particularly immunoglobulins, while facilitating the selective adsorption of tumor-specific proteins. This dual functionality—reduced immune clearance and enhanced tumor targeting—underlies the superior antitumor efficacy observed. The distinct protein adsorption patterns in liver and tumor microenvironments are explained by the different isoelectric points of the proteins and their electrostatic interactions with the functional groups on the CP-LV surface. This suggests a novel strategy for designing nanocarriers that evade immune system clearance while selectively accumulating in tumor sites.

This research introduces a novel strategy for regulating protein corona formation on nanovesicles using glycosylated polyhydroxy polymers. CP₁-LVs, with an optimized amino/hydroxyl ratio, demonstrate significantly improved blood circulation, reduced immunogenicity, enhanced tumor accumulation, and superior antitumor efficacy compared to PEGylated liposomes. This work highlights the potential of rationally designing nanocarrier surfaces to modulate protein corona composition for efficient and targeted drug delivery. Future research should focus on exploring other types of polymers and investigating the long-term effects of CP₁-LVs in various disease models.

This study primarily focused on A549 and HeLa cancer cell lines and their corresponding xenograft models. Further investigations are needed to determine the effectiveness and generalizability of CP₁-LVs in other cancer types and disease models. The long-term toxicity of CP₁-LVs requires further evaluation. The detailed mechanisms of interaction between the modified surface and specific proteins in the corona need to be further explored to optimize the design of surface functional groups.