Juanjuan Du

A novel intracellular protein delivery platform based on single-protein nanocapsules

An average cell contains thousands of proteins that participate in normal cellular functions, and most diseases are somehow related to the malfunctioning of one or more of these proteins. Protein therapy, which delivers proteins into the cell to replace the dysfunctional protein, is considered the most direct and safe approach for treating disease. However, the effectiveness of this method has been limited by its low delivery efficiency and poor stability against proteases in the cell, which digest the protein. Here, we show a novel delivery platform based on nanocapsules consisting of a protein core and a thin permeable polymeric shell that can be engineered to either degrade or remain stable at different pHs. Non-degradable capsules show long-term stability, whereas the degradable ones break down their shells, enabling the core protein to be active once inside the cells. Multiple proteins can be delivered to cells with high efficiency while maintaining low toxicity, suggesting potential applications in imaging, therapy and cosmetics fields.

Biomimetic enzyme nanocomplexes and their use as antidotes and preventive measures for alcohol intoxication

Organisms have sophisticated subcellular compartments containing enzymes that function in tandem. These confined compartments ensure effective chemical transformation and transport of molecules, and the elimination of toxic metabolic wastes. Creating functional enzyme complexes that are confined in a similar way remains challenging. Here we show that two or more enzymes with complementary functions can be assembled and encapsulated within a thin polymer shell to form enzyme nanocomplexes. These nanocomplexes exhibit improved catalytic efficiency and enhanced stability when compared with free enzymes. Furthermore, the co-localized enzymes display complementary functions, whereby toxic intermediates generated by one enzyme can be promptly eliminated by another enzyme. We show that nanocomplexes containing alcohol oxidase and catalase could reduce blood alcohol levels in intoxicated mice, offering an alternative antidote and prophylactic for alcohol intoxication.

Switch-mediated activation and retargeting of CAR-T cells for B-cell malignancies.

Significance Chimeric antigen receptor T (CAR-T) cell therapy has produced promising results in clinical trials but has been challenged by the inability to control engineered cells once infused into the patient. Here we present a generalizable method of controlling CAR-T cells using peptide-engrafted antibody-based molecular switches that act as a bridge between the target cell and CAR-T cell. We show that switches specific for CD19 govern the activity, tissue-homing, cytokine release, and phenotype of switchable CAR-T cells in a dose-titratable manner using xenograft mouse models of B-cell leukemia. We expect that this method of tuning CAR-T cell responses will provide improved safety and versatility of CAR–T-cell therapy in the clinic. Chimeric antigen receptor T (CAR-T) cell therapy has produced impressive results in clinical trials for B-cell malignancies. However, safety concerns related to the inability to control CAR-T cells once infused into the patient remain a significant challenge. Here we report the engineering of recombinant antibody-based bifunctional switches that consist of a tumor antigen-specific Fab molecule engrafted with a peptide neo-epitope, which is bound exclusively by a peptide-specific switchable CAR-T cell (sCAR-T). The switch redirects the activity of the bio-orthogonal sCAR-T cells through the selective formation of immunological synapses, in which the sCAR-T cell, switch, and target cell interact in a structurally defined and temporally controlled manner. Optimized switches specific for CD19 controlled the activity, tissue-homing, cytokine release, and phenotype of sCAR-T cells in a dose-titratable manner in a Nalm-6 xenograft rodent model of B-cell leukemia. The sCAR–T-cell dosing regimen could be tuned to provide efficacy comparable to the corresponding conventional CART-19, but with lower cytokine levels, thereby offering a method of mitigating cytokine release syndrome in clinical translation. Furthermore, we demonstrate that this methodology is readily adaptable to targeting CD20 on cancer cells using the same sCAR-T cell, suggesting that this approach may be broadly applicable to heterogeneous and resistant tumor populations, as well as other liquid and solid tumor antigens.

Controlled Protein Delivery Based on Enzyme‐Responsive Nanocapsules

Enzyme-responsive protein nanocapsules are synthesized to release their protein cargoes in response to specific enzymes secreted in certain cellular events not only with specificity but also with controlled rate by composition tuning. The unique nanocapsule structures protect the encapsulated proteins with robustness against reacting reaction system, providing a new direction towards responsive protein delivery according to specific cellular events or local environment.

Construction of Robust Enzyme Nanocapsules for Effective Organophosphate Decontamination, Detoxification, and Protection

Nanocapsules of organophosphorous hydrolase with enhanced enzyme activity and stability are prepared via in situ polymerization, providing a novel class of nanoparticles for the decontamination and detoxification of organophosphates such as chemical warfare agents and pesticides. Using the nanocapsules as building blocks, bioactive nanocomposites are also fabricated, enabling their use for organophosphate protection.

Quantum-Dot-Decorated Robust Transductable Bioluminescent Nanocapsules

Bioluminescence, due to its high sensitivity, has been exploited in various analytical and imaging applications. In this work, we report a highly stable, cell-transductable, and wavelength-tunable bioluminescence system achieved with an elegant and simple design. Using aqueous in situ polymerization on a bioluminescent enzyme anchored with polymerizable vinyl groups, we obtained nanosized core-shell nanocapsules with the enzyme as the core and a cross-linked thin polymer net as the shell. These nanocapsules possess greatly enhanced stability, retained bioactivity, and a readily engineered surface. In particular, by incorporating polymerizable amines in the polymerization, we endowed the nanocapsules with efficient cell-transduction and sufficient conjugation sites for follow-up modification. Following in situ polymerization, decorating the polymer shell with fluorescent quantum dots allowed us to access a continuous tunable wavelength, which extends the application of such bioluminescent nanocapsules, especially in deep tissue. In addition, the unique core-shell structure and adequate conjugation sites on surface enabled us to maximize the BRET efficiency by adjusting the QD/enzyme conjugation ratio.

Synthetic Nanocarriers for Intracellular Protein Delivery

Introducing exogenous proteins intracellularly presents tremendous chances in scientific research and clinical applications. The effectiveness of this method, however, has been limited by lack of efficient ways to achieve intracellular protein delivery and poor stability of the delivered proteins. Over the years, a variety of nanomaterials have been explored as intracellular protein delivery vectors, including liposomes, polymers, gold nanoparticles, mesoporous silica particles, and carbon nanotubes. Nanomaterials stand out in various protein delivery systems due to various advantages, such as efficient intracellular delivery, long circulation time, and passive tumor targeting. Additionally, chemistry behind these nanomaterials provides readily engineered materials, enabling versatile designs of delivery agents. Intracellular delivery mediated by such nanocarriers achieved varying degrees of success. Different problems associated with these nanocarriers, however, still hamper their real-world applications. Developing new delivery methods or vectors remains essential but challenging. This review surveys the current developments in protein delivery based on synthetic nanocarriers, including liposomes, polymers and inorganic nanocarriers; Prospects for future development of protein delivery nanocarriers are also provided.

Protein-polymer nanoparticles for nonviral gene delivery.

Protein-polymer conjugates were investigated as nonviral gene delivery vectors. BSA-poly(dimethylamino) ethyl methacrylate (PDMA) nanoparticles (nBSA) were synthesized using in situ atom transfer radical polymerization (in situ ATRP) and BSA as a macroinitiator. The diameter and charge of nBSA was a function of the ATRP reaction time and ranged from 5 to 15 nm and +8.9 to +22.5, respectively. nBSA were able to condense plasmid DNA (pDNA) and form polyplexes with an average diameter of 50 nm. nBSA/pDNA polyplexes transfected cells with similar efficiencies or better as compared to linear and branched PEI. Interestingly, the nBSA particle diameter and charge did not affect pDNA complexation and transgene expression, indicating that the same gene delivery efficiency can be achieved with lower charge ratios. We believe that with the use of protein-polymer conjugates additional functionality could be introduced to polyplexes by using different protein cores and, thus, they pose an interesting alternative to the design of nonviral gene delivery vectors.

Mixed Monolayers of Ferrocenylalkanethiol and Encapsulated Horseradish Peroxidase for Sensitive and Durable Electrochemical Detection of Hydrogen Peroxide

This paper describes the construction of a mixed monolayer of ferrocenylalkanethiol and encapsulated horseradish peroxidase (HRP) at a gold electrode for amperometric detection of H(2)O(2) at trace levels. By tuning the alkanethiol chain lengths that tether the HRP enzyme and the ferrocenylalkanethiol (FcC(11)SH) mediator, facile electron transfer between FcC(11)SH and HRP can be achieved. Unlike most HRP-based electrochemical sensors, which rely on HRP-facilitated H(2)O(2) reduction (to H(2)O), the electrocatalytic current is resulted from an HRP-catalyzed oxidation reaction of H(2)O(2) (to O(2)). Upon optimizing other experimental conditions (surface coverage ratio, pH, and flow rate), the electrocatalytic reaction proceeding at the electrode was used to attain a low amperometric detection level (0.64 nM) and a dynamic range spanning over 3 orders of magnitude. Not only does the thin hydrophilic porous HRP capsule allow facile electron transfer, it also enables H(2)O(2) to permeate. More significantly, the enzymatic activity of the encapsulated HRP is retained for a considerably longer period (>3 weeks) than naked HRP molecules attached to an electrode or those wired to a redox polymer thin film. By comparing to electrodes modified with denatured HRP that are subsequently encapsulated or embedded in a poly-L-lysine matrix, it is concluded that the encapsulation has significantly preserved the native structure of HRP and therefore its enzymatic activity. The electrode covered with FcC(11)SH and encapsulated HRP is shown to be capable of rapidly and reproducibly detecting H(2)O(2) present in complex sample media.

A High-Throughput Platform for Formulating and Screening Multifunctional Nanoparticles Capable of Simultaneous Delivery of Genes and Transcription Factors

Simultaneous delivery of multiple genes and proteins (e.g., transcription factors; TFs) is an emerging issue surrounding therapeutic research due to their ability to regulate cellular circuitry. Current gene and protein delivery strategies, however, are based on slow batch synthesis, which is ineffective, poorly controlled, and incapable of simultaneous delivery of both genes and proteins with synergistic functions. Consequently, advances in this field have been limited to in vitro studies. Here, by integrating microfluidic technologies with a supramolecular synthetic strategy, we present a high-throughput approach for formulating and screening multifunctional supramolecular nanoparticles (MFSNPs) self-assembled from a collection of functional modules to achieve simultaneous delivery of one gene and TF with unprecedented efficiency both in vitro and in vivo. We envision that this new approach could open a new avenue for immunotherapy, stem cell reprogramming, and other therapeutic applications.