Previous Research

Biomaterials Drug Delivery Cell Engineering Systems Biology Bioimaging

Biomaterials

ELPs (elastin-like polypeptides) are biopolymers composed of repeating pentapeptide XGVPG and exhibit low critical solution temperature (LCST) phase transition behavior. They are soluble at temperatures below their inverse transition temperature (Tt) and coacervate into insoluble aggregates above their Tt. A short peptide can be easily attached recombinantly to the ELP diblock polymer with different Tt, and they self-assemble into peptide-modified micelles. Thermo-responsive ELP micelles can be used as a nanoparticle platform to facilitate the study of peptide ligand-directed targeted nanoparticle drug delivery. (Wang J, et al. Nano Lett., 2019. My research work in Chilkoti Lab at Duke University.)

Cargo-free porous PCL (polycaprolactone) scaffolds implanted in the breast cancer animal models can mimic pre-metastatic lungs to recruit lung-tropic metastatic cancer cells from the circulation. More importantly, scaffold implants develop potent antitumor immunity resulting from foreign body response (FBR), in contrast to immune-suppressive lungs. Cancer cells seeding the scaffold implants and those seeding the lungs thereafter develop tumor dormancy and outgrowth, respectively. The engineered metastatic niche (polymeric scaffolds) can serve as a model to study immune-modulation in cancer to develop FBR-inspired cancer immunotherapy. (Wang J, et al. BioRxiv, 2020. My research work in Shea Lab at the University of Michigan.)

PEGylated lipids are FDA-approved nonionic diblock copolymers with broad applications in nanomedicine. We found that PEGylated lipid nanoparticles preferentially accumulate in the endoplasmic reticulum (ER) and induce ER stress. Interestingly, cancer cells and normal cells respond differently to biomaterial-induced ER stress. In cancer cells, PEGylated lipids damage ER functions and trigger apoptosis by activating proapoptotic UPR and overproducing cell death effector CHOP. By contrast, in normal cells, high expression of the UPR feedback protein GADD34 triggers antiapoptotic UPR, protecting cells from apoptosis. This study demonstrated PEGlyated lipid safety and implied their potential to synergize with chemotherapy in drug delivery. (Wang J, et al. ACS Nano, 2012. My research work in Liang Lab in the Chinese Academy of Sciences.)

Drug Delivery

The nanoparticle drug delivery system can enhance the accumulation of chemotherapy drugs in the tumor through enhanced penetration and retention (EPR). When doxorubicin (Dox) was physically loaded into PEGylated lipid (PEG-PE) micelles or chemically conjugated to the core of elastin-like polypeptide (ELP) micelles, their circulation time in blood and their accumulation amount in the primary tumor increased compared to free drugs. Besides, tumor cells took up more nanoparticle-delivered doxorubicin that enters cells through efficient endocytosis than free small-molecular drugs that diffuse into tumor cells slowly. Therefore, nanoparticle-delivered doxorubicin achieved higher therapeutic efficacy than free doxorubicin. (Tang N, et al. J Natl. Cancer Inst. 2007; MacKay JA, et al, Nat. Mater., 2009; Wang J, et al. J. Control. Release, 2012; Wang J, et al, J. Control. Release, 2017. My research work in Chilkoti Lab and Liang Lab.)

Many homing peptide ligands are hydrophobic and cannot be presented appropriately at the nanoparticle's surface, resulting in limited accessibility to receptors and sub-optimal targeting. To address this issue, we recombinantly synthesized a fusion protein that included an elastin-like polypeptide diblock copolymer (ELP) carrier, an intervening peptide linker, and a hydrophobic ErbB2-targeted peptide ligand. These polypeptides self-assemble into micelle nanoparticles in the aqueous solution, but hydrophobic ligands would be buried in the micellar core if the micellar surface microenvironment is inappropriate for ligand presentation. After testing more than twenty designs of peptide linkers varied in sequence, length, and charge, we identified the optimal ones that can assist ligands in targeting ErbB2 receptors (a breast cancer marker). Results indicate enhanced uptake of ligand-modified polypeptide micelles in cancer cells after surface modification with optimal linkers. (Wang J, et al. Nano Lett., 2017. My research work in Chilkoti Lab.)

Intratympanic delivery of neurotrophin-3 (NT-3) growth factor can reverse sensorineural hearing loss. But the current poloxamer hydrogel delivery system needs a thermo-controlled injection and has poor mechanical properties and a short retention time. We identified a biocompatible injectable PEG hydrogel superior to poloxamer in terms of shelf time, ease of gel handling, mechanic properties, and in vivo retention time. We also conjugated PEG hydrogels with NT-3 binding peptides screened and identified through the phage display peptide library. The reversible interactions between peptides and NT-3 allow affinity-controlled release, resulting in a 5-fold slower release rate than poloxamer in vivo.  (Wang J, et al. J. Control. Release, 2021. My research work in Shea Lab.)

Cell Engineering

Using the retroviral transfection technique, we developed a genetically engineered MCF-7/Tet-On/ZsGreen-ErbB2 breast cancer cell line in which: (1) the ErbB2 receptor density on the cell membrane can be easily induced and precisely tailored by the doxycycline concentration, spanning from 0.42E5 per cell (ErbB2-negative) to 6.57E5 per cell (ErbB2-positive); and (2) the location and extent of ErbB2 expression can be visualized and measured using the fluorescent ZsGreen tag. With this cell platform, we studied the influence of receptor-ligand multivalent interactions on the cellular uptake of targeted nanoparticles. After testing the interactions at five ligand densities and eleven receptor densities, we identified a matching pattern in which a receptor-to-ligand density ratio of 0.7-4.5 and a minimum of 1.6 bonds are required to initiate receptor-mediated endocytosis of ligand-modified ErbB2-targeted nanoparticles. This study inspires how to design targeted drug delivery rationally.  (Wang J, et al. ACS Nano, 2020. My research work in Chilkoti Lab.)

Shea Lab at the University of Michigan developed a technique termed TRanscriptional Activity CEll aRray (TRACER) that utilizes lentiviral transfection to develop engineered cells capable of producing luciferase-based luminescence signals when transcription factors (TFs) inside cells are overexpressed. TRACER provides a quantitative measure of the relative transactivation of TFs in live cells and can also measure the large-scale dynamic activity of multiple TFs. We applied this technique to study the environmental regulation of tumor dormancy and outgrowth. We identified activation of different signaling pathways in cancer cells in response to signals from the engineered metastatic niche environment with potent antitumor immunity (scaffold implants in cancer models) and an immunosuppressive protumor environment in metastatic lungs. (Decker JT, et al. Biotechnol Bioeng, 2017; Wang J, et al. Submitted. 2021. My research work in Shea Lab.)

Systems Biology

TRACER allows a large-scale dynamic analysis of transcription factors (TFs). OpenArray and single-cell RNA sequencing (scRNA-seq) allow a large-scale analysis of gene expression. CytokineArray allows a large-scale analysis of secreted proteins. We utilized these techniques and computational analysis to study the mechanism by which the foreign body responses (FBR) reverse cancer-induced immunosuppressive signals and create an immune-active environment with high antitumor immunity in the engineered metastatic niche (scaffold implants in cancer models). (Wang J, et al. Submitted, 2021. My research work in Shea Lab.)

Bioimaging

We developed a method that combines ratiometric imaging with quantitative image analysis, which allows the quantification of endo-lysosomal pH in live cells. This method treated cells with Lysosensor Yellow/Blue pH indicator that accumulated in endo-lysosomes and exhibited dual pH-dependent emission peaks at 440 nm (blue fluorescence) and 530 nm (green fluorescence). Lysosensor-treated cells were imaged by a spinning disk confocal microscope equipped with EMCCD at blue and green channels. Images with a resolution of 512 pixels × 512 pixels were analyzed by ImageJ and MATLAB. Every pixel's Blue/Green ratio was converted to a pH value and the spatial endo-lysosomal pH in live cells was mapped. This method compared the cellular distribution of untargeted nanoparticles and targeted nanoparticles modified with cell-penetrating peptides (CPPs). We also studied the subcellular trafficking dynamics of nanoparticles loaded with fluorescent doxorubicin.  (Wang J, et al. Nano Lett., 2016; Wang J, et al. J. Control. Release. 2017. My research work in Chilkoti Lab.)

Using the fluorescence resonance energy transfer (FRET) technique, we studied the internalization of AF650-labeled polymeric micelle nanoparticles physically encapsulated with fluorescent doxorubicin (Dox) drugs. When doxorubicin is released from the micellar core, FRET from doxorubicin to AF650 disappears and doxorubicin's fluorescence recovers. With this method, we identified polymeric micelles disassemble at the cell surface upon polymers inserting the cell membrane, and then drugs and carriers enter cells separately. (Wang J, et al. J. Control. Release, 2012. My research work in Liang Lab.)

Using the fluorescence de-quenching technique, we studied the intracellular trafficking of BHQ2-labeled polypeptide micelle nanoparticles chemically conjugated with doxorubicin by pH-sensitive linkers. BHQ2 quenches doxorubicin in the micellar core. When doxorubicin is released from micelles upon acid-triggered linker cleavage, doxorubicin separates from BHQ2 and recovers its fluorescence. This method demonstrated that pH-sensitive linkers are an efficient strategy to control drug release in lysosomes. (Wang J, et al. J. Control. Release. 2017. My research work in Chilkoti Lab.)

We prepared a dual-fluorescent drug-loaded polypeptide micelle nanoparticle with pH-responsive activity and studied its distribution and acid-triggered drug release in vivo. Fluorescent doxorubicin was conjugated to the polymer via a pH-sensitive hydrazone linker, and the AF750 fluorophore was attached to the polymer via a pH-insensitive amide linker. We administrated the dual-fluorescent nanoparticle to tumor-bearing mice through i.v. injection, and monitored the whole-body fluorescence by IVIS imaging system. We observed that AF750-labeled polymer reached maximum accumulation in the kidney at ~10 h after injection and in the tumor at ~24 h after injection (EPR effects). Consistently, the accumulation of doxorubicin in the kidney was not evident due to the fast elimination of nanoparticles, but drug accumulation in the tumor was conspicuous. The fluorescent intensity ratio of doxorubicin to AF750 reflects the drug release as a result of acid-triggered cleavage of hydrazone linkers. The increased ratio in the tumor suggests the continuous linker cleavage and drug release in the tumor triggered by low pH, and maximum drug release occurred at ~80 h after injection. (Wang J, et al. 2015. My research work in Chilkoti Lab.)