Protein nanoparticles as drug delivery systems for cancer theranostics.

Protein-based nanoparticles have garnered significant attention in theranostic applications due to their superior biocompatibility, exceptional biodegradability and ease of functionality. Compared to other nanocarriers, protein-based nanoparticles offer additional advantages, including biofunctionality and precise molecular recognition abilities, which make them highly effective in navigating complex biological environments. Moreover, proteins can serve as powerful tools with self-assembling structures and reagents that enhance cell penetration. And their derivation from abundant renewable sources and ability to degrade into harmless amino acids further enhance their suitability for biomedical applications. However, protein-based nanoparticles have so far not realized their full potential. In this review, we summarize recent advances in the use of protein nanoparticles in tumor diagnosis and treatment and outline typical methods for preparing protein nanoparticles. The review of protein nanoparticles may provide useful new insights into the development of biomaterial fabrication.

Accelerating diabetic wound healing by ROS-scavenging lipid nanoparticle-mRNA formulation.

Current treatment options for diabetic wounds face challenges due to low efficacy, as well as potential side effects and the necessity for repetitive treatments. To address these issues, we report a formulation utilizing trisulfide-derived lipid nanoparticle (TS LNP)-mRNA therapy to accelerate diabetic wound healing by repairing and reprogramming the microenvironment of the wounds. A library of reactive oxygen species (ROS)-responsive TS LNPs was designed and developed to encapsulate interleukin-4 (IL4) mRNA. TS2-IL4 LNP-mRNA effectively scavenges excess ROS at the wound site and induces the expression of IL4 in macrophages, promoting the polarization from the proinflammatory M1 to the anti-inflammatory M2 phenotype at the wound site. In a diabetic wound model of db/db mice, treatment with this formulation significantly accelerates wound healing by enhancing the formation of an intact epidermis, angiogenesis, and myofibroblasts. Overall, this TS LNP-mRNA platform not only provides a safe, effective, and convenient therapeutic strategy for diabetic wound healing but also holds great potential for clinical translation in both acute and chronic wound care.

Engineering LNPs with polysarcosine lipids for mRNA delivery.

Since the approval of the lipid nanoparticles (LNP)-mRNA vaccines against the SARS-CoV-2 virus, there has been an increased interest in the delivery of mRNA through LNPs. However, current LNP formulations contain PEG lipids, which can stimulate the generation of anti-PEG antibodies. The presence of these antibodies can potentially cause adverse reactions and reduce therapeutic efficacy after administration. Given the widespread deployment of the COVID-19 vaccines, the increased exposure to PEG may necessitate the evaluation of alternative LNP formulations without PEG components. In this study, we investigated a series of polysarcosine (pSar) lipids as alternatives to the PEG lipids to determine whether pSar lipids could still provide the functionality of the PEG lipids in the ALC-0315 and SM-102 LNP systems. We found that complete replacement of the PEG lipid with a pSar lipid can increase or maintain mRNA delivery efficiency and exhibit similar safety profiles in vivo.

Redox-responsive polymer micelles co-encapsulating immune checkpoint inhibitors and chemotherapeutic agents for glioblastoma therapy.

Immunotherapy with immune checkpoint blockade (ICB) for glioblastoma (GBM) is promising but its clinical efficacy is seriously challenged by the blood-tumor barrier (BTB) and immunosuppressive tumor microenvironment. Here, anti-programmed death-ligand 1 antibodies (aPD-L1) are loaded into a redox-responsive micelle and the ICB efficacy is further amplified by paclitaxel (PTX)-induced immunogenic cell death (ICD) via a co-encapsulation approach for the reinvigoration of local anti-GBM immune responses. Consequently, the micelles cross the BTB and are retained in the reductive tumor microenvironment without altering the bioactivity of aPD-L1. The ICB efficacy is enhanced by the aPD-L1 and PTX combination with suppression of primary and recurrent GBM, accumulation of cytotoxic T lymphocytes, and induction of long-lasting immunological memory in the orthotopic GBM-bearing mice. The co-encapsulation approach facilitating efficient antibody delivery and combining with chemotherapeutic agent-induced ICD demonstrate that the chemo-immunotherapy might reprogram local immunity to empower immunotherapy against GBM.

LNP-RNA-engineered adipose stem cells for accelerated diabetic wound healing.

Adipose stem cells (ASCs) have attracted considerable attention as potential therapeutic agents due to their ability to promote tissue regeneration. However, their limited tissue repair capability has posed a challenge in achieving optimal therapeutic outcomes. Herein, we conceive a series of lipid nanoparticles to reprogram ASCs with durable protein secretion capacity for enhanced tissue engineering and regeneration. In vitro studies identify that the isomannide-derived lipid nanoparticles (DIM1T LNP) efficiently deliver RNAs to ASCs. Co-delivery of self-amplifying RNA (saRNA) and E3 mRNA complex (the combination of saRNA and E3 mRNA is named SEC) using DIM1T LNP modulates host immune responses against saRNAs and facilitates the durable production of proteins of interest in ASCs. The DIM1T LNP-SEC engineered ASCs (DS-ASCs) prolong expression of hepatocyte growth factor (HGF) and C-X-C motif chemokine ligand 12 (CXCL12), which show superior wound healing efficacy over their wild-type and DIM1T LNP-mRNA counterparts in the diabetic cutaneous wound model. Overall, this work suggests LNPs as an effective platform to engineer ASCs with enhanced protein generation ability, expediting the development of ASCs-based cell therapies.

Adoptive cell therapy for cancer treatment.

Adoptive cell therapy (ACT) is a rapidly growing anti-cancer strategy that has shown promise in treating various cancer types. The concept of ACT involves activating patients' own immune cells ex vivo and then transferring them back to the patients to recognize and eliminate cancer cells. Currently, the commonly used ACT includes tumor-infiltrating lymphocytes (TILs), genetically engineered immune cells, and dendritic cells (DCs) vaccines. With the advancement of cell culture and genetic engineering techniques, ACT has been used in clinics to treat malignant hematological diseases and many new ACT-based regimens are in different stages of clinical trials. Here, representative ACT approaches are introduced and the opportunities and challenges for clinical translation of ACT are discussed.

Close the cancer-immunity cycle by integrating lipid nanoparticle-mRNA formulations and dendritic cell therapy.

Effective cancer immunotherapy is usually blocked by immunosuppressive factors in the tumour microenvironment, resulting in tumour promotion, metastasis and recurrence. Here we combine lipid nanoparticle-mRNA formulations and dendritic cell therapy (named CATCH) to boost the cancer-immunity cycle via progressive steps to overcome the immunosuppressive tumour microenvironment. Multiple types of sugar-alcohol-derived lipid nanoparticles are conceived to modulate the cancer-immunity cycle. First, one type of lipid nanoparticle containing CD40 ligand mRNA induces robust immunogenic cell death in tumoural tissues, leading to the release of tumour-associated antigens and the expression of CD40 ligand. Next, dendritic cells engineered by another type of lipid nanoparticle encapsulating CD40 mRNA are adoptively transferred, which are then activated by the CD40 ligand molecules in tumoural tissues. This promotes the secretion of multiple cytokines and chemokines, and the upregulation of co-stimulatory molecules on dendritic cells, which are crucial for reprogramming the tumour microenvironment and priming the T-cell responses. After dendritic cells present tumour-associated antigens to T cells, all the above stepwise events contribute to boosting a potent tumour-specific T-cell immunity that eradicates established tumours, suppresses distal lesions and prevents tumour rechallenge.

Recruiting T-Cells toward the Brain for Enhanced Glioblastoma Immunotherapeutic Efficacy by Co-Delivery of Cytokines and Immune Checkpoint Antibodies with Macrophage-Membrane-Camouflaged Nanovesicles.

Immunotherapy with immune checkpoint inhibitors (CPIs) shows promising prospects for glioblastoma multiforme (GBM) but with restricted results, mainly attributed to the immunosuppressive tumor microenvironment (TME) and the limited antibody permeability of the blood-tumor barrier (BTB) in GBM. Here, nanovesicles with a macrophage-mimicking membrane are described, that co-deliver chemotactic CXC chemokine ligand 10 (CXCL10), to pre-activate the immune microenvironment, and anti-programmed death ligand 1 antibody (aPD-L1), to interrupt the immune checkpoint, aiming to enhance the effect of GBM immunotherapy. Consequently, the tumor tropism of the macrophage membrane and the receptor-mediated transcytosis of the angiopep-2 peptide allow the nanovesicle to effectively cross the BTB and target the GBM region, with 19.75-fold higher accumulation of antibodies compared to the free aPD-L1 group. The CPI therapeutic efficacy is greatly enhanced by CXCL10-induced T-cells recruitment with significant expansion of CD8+ T-cells and effector memory T-cells, leading to the elimination of tumor, prolonged survival time, and long-term immune memory in orthotopic GBM mice. The nanovesicles, that relieve the tumor immunosuppressive microenvironment by CXCL10 to enhance aPD-L1 efficacy, may present a promising strategy for brain-tumor immunotherapy.

Advances in engineering and delivery strategies for cytokine immunotherapy.

INTRODUCTION: Cytokine immunotherapy is a growing field for the treatment of cancer, infectious disease, autoimmunity, and other ailments. Therapeutic cytokines are a class of secreted, small proteins that play a pivotal role in regulating the innate and adaptive immune system by provoking or mitigating immune responses. In the clinic, cytokines are frequently combined with other treatments, such as small molecules and monoclonal antibodies. However, the clinical translation of cytokine therapies is hindered by their short half-life, pleiotropic nature, and off-target effects, which cause diminished efficacy and severe systemic toxicity. Such toxicity limits dosage, thus resulting in suboptimal doses. Accordingly, numerous efforts have been devoted to exploring strategies to promote cytokine therapies by improving their tissue specificity and pharmacokinetics. AREAS COVERED: Preclinical and clinical research into bioengineering and delivery strategies for cytokines, consisting of bioconjugation, fusion proteins, nanoparticles, and scaffold-based systems. EXPERT OPINION: These approaches pave the way for the development of next-generation cytokine treatments with greater clinical benefit and reduced toxicity, circumventing such issues currently associated with cytokine therapy.

STING Agonist-Derived LNP-mRNA Vaccine Enhances Protective Immunity Against SARS-CoV-2.

Lipid nanoparticle (LNP)-mediated delivery of messenger RNA (mRNA) COVID-19 vaccines has provided large-scale immune protection to the public. To elicit a robust immune response against SARS-CoV-2 infections, antigens produced by mRNAs encoding SARS-CoV-2 Spike glycoprotein need to be efficiently delivered and presented to antigen-presenting cells such as dendritic cells (DCs). As concurrent innate immune stimulation can facilitate the antigen presentation process, a library of non-nucleotide STING agonist-derived amino lipids (SALs) was synthesized and formulated into LNPs for mRNA delivery. SAL12 lipid nanoparticles (SAL12-LNPs) were identified as most potent in delivering mRNAs encoding the Spike glycoprotein (S) of SARS-CoV-2 while activating the STING pathway in DCs. Two doses of SAL12 S-LNPs by intramuscular immunization elicited potent neutralizing antibodies against SARS-CoV-2 in mice.

Cholesterol-Amino-Phosphate (CAP) Derived Lipid Nanoparticles for Delivery of Self-Amplifying RNA and Restoration of Spermatogenesis in Infertile Mice.

Male infertility caused by genetic mutations is an important type of infertility. Currently, there is no reliable method in the clinic to address this medical need. The emergence of mRNA therapy provides a possible strategy for restoring mutant genes in the reproductive system. However, effective delivery of mRNA to spermatocytes remains a formidable challenge. Here a series of cholesterol-amino-phosphate (CAP) lipids are reported by integrating three bioactive moieties into a geometric structure, which is favorable for mRNA delivery. The results demonstrate that CAP-derived lipid nanoparticles (CAP LNPs) can deliver RNA including traditional mRNA and self-amplifying RNA (saRNA) encoding DNA Meiotic Recombinase 1 (Dmc1) protein in spermatocytes and treat male infertility caused by the Dmc1 gene mutation. Notably, the delivery efficiency of CAP LNPs is significantly higher than that of the MC3 and ALC-0315 LNPs, which is consistent with the design of CAP molecules. More importantly, a single injection of CAP LNPs-saRNA can produce Dmc1 protein for an extended period, which restores the spermatogenesis in the Dmc1 gene knockout mouse model. Overall, this study proves the concept of LNPs for the delivery of mRNA to spermatocytes, which provides a unique method to probe male infertility caused by the genetic mutation.

Nanomaterials-Mediated Co-Stimulation of Toll-Like Receptors and CD40 for Antitumor Immunity.

Toll-like receptors (TLRs) and CD40-related signaling pathways represent critical bridges between innate and adaptive immune responses. Here, an immunotherapy regimen that enables co-stimulation of TLR7/8- and CD40-mediated pathways is developed. TLR7/8 agonist resiquimod (R848) derived amino lipids, RAL1 and RAL2, are synthesized and formulated into RAL-derived lipid nanoparticles (RAL-LNPs). The RAL2-LNPs show efficient CD40 mRNA delivery to DCs both in vitro (90.8 ± 2.7%) and in vivo (61.3 ± 16.4%). When combined with agonistic anti-CD40 antibody, this approach can produce effective antitumor activities in mouse melanoma tumor models, thereby suppressing tumor growth, prolonging mouse survival, and establishing antitumor memory immunity. Overall, RAL2-LNPs provide a novel platform toward cancer immunotherapy by integrating innate and adaptive immunity.

Recent advances in biomaterial-boosted adoptive cell therapy.

Adoptive immunotherapies based on the transfer of functional immune cells hold great promise in treating a wide range of malignant diseases, especially cancers, autoimmune diseases, and infectious diseases. However, manufacturing issues and biological barriers lead to the insufficient population of target-selective effector cells at diseased sites after adoptive transfer, hindering effective clinical translation. The convergence of immunology, cellular biology, and materials science lays a foundation for developing biomaterial-based engineering platforms to overcome these challenges. Biomaterials can be rationally designed to improve ex vivo immune cell expansion, expedite functional engineering, facilitate protective delivery of immune cells in situ, and navigate the infused cells in vivo. Herein, this review presents a comprehensive summary of the latest progress in biomaterial-based strategies to enhance the efficacy of adoptive cell therapy, focusing on function-specific biomaterial design, and also discusses the challenges and prospects of this field.

Recent advances of biomaterials in stem cell therapies.

Stem cells have been utilized as 'living drugs' in clinics for decades. Their self-renewal, differentiation, and immunomodulating properties provide potential solutions for a variety of malignant diseases and disorders. However, the pathological environment may diminish the therapeutic functions and survival of the transplanted stem cells, causing failure in clinical translation. To overcome these challenges, researchers have developed biomaterial-based strategies that facilitatein vivotracking, functional engineering, and protective delivery of stem cells, paving the way for next-generation stem cell therapies. In this perspective, we briefly overview different types of stem cells and the major clinical challenges and summarize recent progress of biomaterials applied to boost stem cell therapies.

Construction of Messenger RNA (mRNA) Probes Delivered By Lipid Nanoparticles to Visualize Intracellular Protein Expression and Localization at Organelles.

Organelles are specialized compartments, where various proteins reside and play crucial roles to maintain essential cellular structures and functions in mammalian cells. A comprehensive understanding of protein expressions and subsequent localizations at each organelle is of great benefit to the development of organelle-based therapies. Herein, a set of single or dual organelle labeling messenger RNAs (SOLAR or DOLAR) is designed as novel imaging probes, which encode fluorescent proteins with various organelle localization signals. These mRNA probes enable to visualize the protein localizations at different organelles and investigate their trafficking from ribosomal machinery to specific organelles. According to the in vitro results, SOLAR probes show organelle targeting capabilities consistent with the design. Moreover, DOLAR probes with different linkers display distinct targeting properties depending on different organelle localization signals. Additionally, these mRNA probes also exhibit organelle labeling ability in vivo when delivered by lipid nanoparticles (LNPs). Therefore, these mRNA-based probes provide a unique tool to study cell organelles and may facilitate the design of organelle-based therapies.

Dual-Sensitive Nanomicelles Enhancing Systemic Delivery of Therapeutically Active Antibodies Specifically into the Brain.

Delivering therapeutic antibodies into the brain across the blood-brain barrier at a therapeutic level is a promising while challenging approach in the treatment of neurological disorders. Here, we present a polymeric nanomicelle (PM) system capable of delivering therapeutically effective levels of 3D6 antibody fragments (3D6-Fab) into the brain parenchyma for inhibiting Aß aggregation. PM assembly was achieved by charge-converting 3D6-Fab through pH-sensitive citraconylation to allow complexation with reductive-sensitive cationic polymers. Brain targeting was achieved by functionalizing the PM surface with glucose molecules to allow interaction with recycling glucose transporter (Glut)-1 proteins. Consequently, 41-fold enhanced 3D6-Fab accumulation in the brain was achieved by using the PM system compared to free 3D6-Fab. Furthermore, therapeutic benefits were obtained by successfully inhibiting Aß1-42 aggregation in Alzheimer's disease mice systemically treated with 3D6-Fab-loaded glucosylated PM. Hence, this nanocarrier system represents a promising method for effectively delivering functional antibody agents into the brain and treating neurological diseases.

Long-term storage of lipid-like nanoparticles for mRNA delivery.

Lipid-like nanoparticles (LLNs) have been extensively explored for messenger RNA (mRNA) delivery in various biomedical applications. However, the long-term storage of these nanoparticles is still a challenge for their clinical translation. In this study, we investigated a series of conditions for the long-term storage of LLNs with encapsulation of mRNA. We evaluated the stability of LLNs with different concentrations of cryoprotectants (sucrose, trehalose or mannitol) under the conditions of freezing or lyophilization processes. Through in vitro and in vivo mRNA delivery studies, we identified the optimal storage condition, and found that the addition with 5% (w/v) sucrose or trehalose to LLNs could remain their mRNA delivery efficiency for at least three months in the liquid nitrogen storage condition.

A Synthetic Carrier of Nucleic Acids Structured as a Neutral Phospholipid Envelope Tightly Assembled on Polyplex Surface.

Synthetic carriers of nucleic acids remain inefficient for practical applications due to their insufficient functions as compared with viral vectors developed by evolution. Here, a synthetic carrier is designed to structurally mimic lentivirus, a widely-used viral vector in therapeutic developments, for its neutral phospholipid membrane tightly anchored on the surface of a packed nucleic acid core. Unlike the reported lipopolyplexes of which the surface membrane around the nucleic acid core is formed from charged lipids, the stable attachment of the neutral lipids to each polyplex core in the present system is achieved through preadsorbed micelles of multicarboxyl amphiphilic molecules as lipid bilayer anchors. The adsorbed micelles are under a tension of deformation due to the electrostatic attraction of the head groups to the cationic surface and their "thermodynamic responsibility" to cover the hydrophobic tails in water. When liposomes of neutral phospholipids approach, the hydrophobic tail groups of the adsorbed micelles may insert into the lipid bilayer matrix to induce them to fuse around polyplex and relieve the thermodynamic tension. The formed neutral phospholipid membrane may encapsulate the polyplex core stably, prevent siRNA from prephagocytic leaking and degrading, and immobilize functional agents with increased capacity.

Comparison of Biological Responses of Polymers Based on Imine and Disulfide Backbones for siRNA Delivery.

To achieve a successful delivery of siRNA by carriers in vivo, the degradation of polymers in response to tiny intracellular changes should be seriously considered. In addition, the balance between degradation and stability of polymers is another key point for high performance of carriers. In this study, imine and disulfide linkages, which are sensitive to pH changes and redox environment, respectively, were constructed as the main backbone of polymers to deliver siRNA at the intracellular and animal level. Comparisons were made between performances of these two different polymers. Both of the polymers synthesized here have good ability to condense siRNA. However, polyplexes formed by the imine backbone-based polymer (TPSP) showed a larger particle size and a higher zeta potential than that of the disulfide backbone-based polymer (DTDPS). Although both TPSP and DTDPS could deliver the target siRNA into 7721 cells, polyplexes formed by TPSP showed a higher silence efficiency in vitro and accomplished more accumulation in tumors. In conclusion, we believe TPSP is superior to be used for siRNA delivery and promises a potential for widespread use.