Rational Design of Nanocarriers for Intracellular Protein Delivery

Protein/antibody therapeutics have exhibited the advantages of high specificity and activity even at an extremely low concentration compared to small molecule drugs. However, they are accompanied by unfavorable physicochemical properties such as fragile tertiary structure, large molecular size, and poor penetration of the membrane, and thus the clinical use of protein drugs is hindered by inefficient delivery of proteins into the host cells. To overcome the challenges associated with protein therapeutics and enhance their biopharmaceutical applications, various protein‐loaded nanocarriers with desired functions, such as lipid nanocapsules, polymeric nanoparticles, inorganic nanoparticles, and peptides, are developed. In this review, the different strategies for intracellular delivery of proteins are comprehensively summarized. Their designed routes, mechanisms of action, and potential therapeutics in live cells or in vivo are discussed in detail. Furthermore, the perspective on the new generation of delivery systems toward the emerging area of protein‐based therapeutics is presented as well.     

Core‐Cone Structured Monodispersed Mesoporous Silica Nanoparticles with Ultra‐large Cavity for Protein Delivery

A new type of monodispersed mesoporous silica nanoparticles with a core–cone structure (MSN‐CC) has been synthesized. The large cone‐shaped pores are formed by silica lamellae closely packed encircling a spherical core, showing a structure similar to the flower dahlia. MSN‐CC has a large pore size of 45 nm and a high pore volume of 2.59 cm³ g⁻¹. MSN‐CC demonstrates a high loading capacity of large proteins and successfully delivers active β‐galactosidase into cells, showing their potential as efficient nanocarriers for the cellular delivery of proteins with large molecular weights.     

Bioinspired Diselenide‐Bridged Mesoporous Silica Nanoparticles for Dual‐Responsive Protein Delivery

Controlled delivery of protein therapeutics remains a challenge. Here, the inclusion of diselenide‐bond‐containing organosilica moieties into the framework of silica to fabricate biodegradable mesoporous silica nanoparticles (MSNs) with oxidative and redox dual‐responsiveness is reported. These diselenide‐bridged MSNs can encapsulate cytotoxic RNase A into the 8–10 nm internal pores via electrostatic interaction and release the payload via a matrix‐degradation controlled mechanism upon exposure to oxidative or redox conditions. After surface cloaking with cancer‐cell‐derived membrane fragments, these bioinspired RNase A‐loaded MSNs exhibit homologous targeting and immune‐invasion characteristics inherited from the source cancer cells. The efficient in vitro and in vivo anti‐cancer performance, which includes increased blood circulation time and enhanced tumor accumulation along with low toxicity, suggests that these cell‐membrane‐coated, dual‐responsive degradable MSNs represent a promising platform for the delivery of bio‐macromolecules such as protein and nucleic acid therapeutics.     

Mesoporous Silica Nanoparticles for Intracellular Controlled Drug Delivery

The application of nanotechnology in the field of drug delivery has attracted much attention in the latest decades. Recent breakthroughs on the morphology control and surface functionalization of inorganic‐based delivery vehicles, such as mesoporous silica nanoparticles (MSNs), have brought new possibilities to this burgeoning area of research. The ability to functionalize the surface of mesoporous‐silica‐based nanocarriers with stimuli‐responsive groups, nanoparticles, polymers, and proteins that work as caps and gatekeepers for controlled release of various cargos is just one of the exciting results reported in the literature that highlights MSNs as a promising platform for various biotechnological and biomedical applications. This review focuses on the most recent progresses in the application of MSNs for intracellular drug delivery. The latest research on the pathways of entry into live mammalian and plant cells together with intracellular trafficking are described. One of the main areas of interest in this field is the development of site‐specific drug delivery vehicles; the contribution of MSNs toward this topic is also summarized. In addition, the current research progress on the biocompatibility of this material in vitro and in vivo is discussed. Finally, the latest breakthroughs for intracellular controlled drug release using stimuli‐responsive mesoporous‐silica‐based systems are described.     

Multifunctional Hybrid Nanoparticles for Traceable Drug Delivery and Intracellular Microenvironment‐Controlled Multistage Drug‐Release in Neurons

Innovative nanoparticles hold promising potential for disease therapy as drug delivery systems. For brain‐disease therapy, a drug delivery system that can sustainably control drug‐release and monitor fluorescence of the drug cargos is highly desirable. In this study, a light‐traceable and intracellular microenvironment‐responsive drug delivery system was developed based on the combination of glutathione‐responsive autoflurescent nanogel, dendrimer‐like mesoporous silica nanoparticles, and gold nanoparticles. The resulting hybrid nanoparticles represent a new class of delivery system that can efficiently load, transport, and control multistage‐release of sulfydryl‐containing drugs into neurons, with light‐traceable monitoring for future brain‐disease therapy.     

Hybrid Protein Nano‐Reactors Enable Simultaneous Increments of Tumor Oxygenation and Iodine‐131 Delivery for Enhanced Radionuclide Therapy

It is hard for current radionuclide therapy to render solid tumors desirable therapeutic efficacy owing to insufficient tumor‐targeted delivery of radionuclides and severe tumor hypoxia. In this study, a biocompatible hybrid protein nanoreactor composed of human serum albumin (HSA) and catalase (CAT) molecules is constructed via glutaraldehyde‐mediated crosslinking. The obtained HSA‐CAT nanoreactors (NRs) show retained and well‐protected enzyme stability in catalyzing the decomposition of H₂O₂ and enable efficient labeling of therapeutic radionuclide iodine‐131 (¹³¹I). Then, it is uncovered that such HSA‐CAT NRs after being intravenously injected into tumor‐bearing mice exhibit efficient passive tumor accumulation as vividly visualized under the fluorescence imaging system and gamma camera. As the result, such HSA‐CAT NRs upon tumor accumulation would significantly attenuate tumor hypoxia by decomposing endogenous H₂O₂ produced by cancer cells to molecular oxygen, and thereby remarkably improve the therapeutic efficacy of radionuclide ¹³¹I. This study highlights the concise preparation of biocompatible protein nanoreactors with efficient tumor homing and hypoxia attenuation capacities, thus enabling greatly improved tumor radionuclide therapy with promising potential for future clinical translation.     

Activatable Protein Nanoparticles for Targeted Delivery of Therapeutic Peptides

Clinical translation of therapeutic peptides, particularly those that require penetration of the cell membrane or are cytolytic, is a major challenge. A novel approach based on a complementary mechanism, which has been widely used for guided synthesis of DNA or RNA nanoparticles, for de novo design of activatable protein nanoparticles (APNPs) for targeted delivery of therapeutic peptides is described. APNPs are formed through self‐assembly of three independent polypeptides based on pairwise coiled‐coil dimerization. They are capable of long circulation in the blood and can be engineered to target diseases. Peptides to be delivered are incorporated into APNPs and released into the disease microenvironment by locally enriched proteases. It is demonstrated that APNPs mediate efficient delivery of NR2B9c, a neuroprotective peptide that functions after cell penetration, and melittin, a cytolytic peptide that perturbs the lipid bilayer, for effective treatment of stroke and cancer, respectively. Due to their robust properties, simple design, and economic costs, APNPs have great potential to serve as a versatile platform for controlled delivery of therapeutic peptides.     

One‐Pot Synthesis of Multiple Protein‐Encapsulated DNA Flowers and Their Application in Intracellular Protein Delivery

Inspired by biological systems, many biomimetic methods suggest fabrication of functional materials with unique physicochemical properties. Such methods frequently generate organic–inorganic composites that feature highly ordered hierarchical structures with intriguing properties, distinct from their individual components. A striking example is that of DNA–inorganic hybrid micro/nanostructures, fabricated by the rolling circle technique. Here, a novel concept for the encapsulation of bioactive proteins in DNA flowers (DNF) while maintaining the activity of protein payloads is reported. A wide range of proteins, including enzymes, can be simultaneously associated with the growing DNA strands and Mg₂PPi crystals during the rolling circle process, ultimately leading to the direct immobilization of proteins into DNF. The unique porous structure of this construct, along with the abundance of Mg ions and DNA molecules present, provides many interaction sites for proteins, enabling high loading efficiency and enhanced stability. Further, as a proof of concept, it is demonstrated that the DNF can deliver payloads of cytotoxic protein (i.e., RNase A) to the cells without a loss in its biological function and structural integrity, resulting in highly increased cell death compared to the free protein.     

A Vesicle Supra‐Assembly Approach to Synthesize Amine‐Functionalized Hollow Dendritic Mesoporous Silica Nanospheres for Protein Delivery

Intracellular delivery of proteins is a promising strategy of intervention in disease, which relies heavily on the development of efficient delivery platforms due to the cell membrane impermeability of native proteins, particularly for negatively charged large proteins. This work reports a vesicle supra‐assembly approach to synthesize novel amine‐functionalized hollow dendritic mesoporous silica nanospheres (A‐HDMSN). An amine silica source is introduced into a water–oil reaction solution prior to the addition of conventional silica source tetraethylorthosilicate. This strategy favors the formation of composite vesicles as the building blocks which further assemble into the final product. The obtained A‐HDMSN have a cavity core of ≈170 nm, large dendritic mesopores of 20.7 nm in the shell and high pore volume of 2.67 cm³ g^?1. Compared to the calcined counterpart without amine groups (C‐HDMSN), A‐HDMSN possess enhanced loading capacity to large negative proteins (IgG and β‐galactosidase) and improved cellular uptake performance, contributed by the cationic groups. A‐HDMSN enhance the intracellular uptake of β‐galactosidase by up to 5‐fold and 40‐fold compared to C‐HDMSN and free β‐galactosidase, respectively. The active form of β‐galactosidase delivered by A‐HDMSN retains its intracellular catalytic functions.     

Synthesis and Assembly of Click‐Nucleic‐Acid‐Containing PEG–PLGA Nanoparticles for DNA Delivery

Co‐delivery of both chemotherapy drugs and siRNA from a single delivery vehicle can have a significant impact on cancer therapy due to the potential for overcoming issues such as drug resistance. However, the inherent chemical differences between charged nucleic acids and hydrophobic drugs have hindered entrapment of both components within a single carrier. While poly(ethylene glycol)‐block‐poly(lactic‐co‐glycolic acid) (PEG–PLGA) copolymers have been used successfully for targeted delivery of chemotherapy drugs, loading of DNA or RNA has been poor. It is demonstrated that significant amounts of DNA can be encapsulated within PLGA‐containing nanoparticles through the use of a new synthetic DNA analog, click nucleic acids (CNAs). First, triblock copolymers of PEG‐CNA‐PLGA are synthesized and then formulated into polymer nanoparticles from oil‐in‐water emulsions. The CNA‐containing particles show high encapsulation of DNA complementary to the CNA sequence, whereas PEG‐PLGA alone shows minimal DNA loading, and non‐complementary DNA strands do not get encapsulated within the PEG‐CNA‐PLGA nanoparticles. Furthermore, the dye pyrene can be successfully co‐loaded with DNA and lastly, a complex, larger DNA sequence that contains an overhang complementary to the CNA can also be encapsulated, demonstrating the potential utility of the CNA‐containing particles as carriers for chemotherapy agents and gene silencers.     

Gold Nanoparticle–Protein Agglomerates as Versatile Nanocarriers for Drug Delivery

The fabrication of a versatile nanocarrier based on agglomerated structures of gold nanoparticle (Au NP)–lysozyme (Lyz) in aqueous medium is reported. The carriers exhibit efficient loading capacities for both hydrophilic (doxorubicin) and hydrophobic (pyrene) molecules. The nanocarriers are finally coated with an albumin layer to render them stable and also facilitate their uptake by cancer cells. The interaction between agglomerated structures and the payloads is non‐covalent. Cell viability assay in vitro showed that the nanocarriers by themselves are non‐cytotoxic, whereas the doxorubicin‐loaded ones are cytotoxic, with efficiencies higher than that of the free drug. Transmission electron microscopy and fluorescence microscopy along with flow cytometry analysis confirm the uptake of the drug‐loaded nanocarriers by a human cervical cancer HeLa cell line. Field‐emission scanning electron microscopy reveals the formation of apoptotic bodies leading to cell death, confirming the release of the payloads from the nanocarriers into the cell. Overall, the findings suggest the fabrication of novel Au NP–protein agglomerate‐based nanocarriers with efficient drug‐loading and ‐releasing capabilities, enabling them to act as multimodal drug‐delivery vehicles.     

Immune Cell‐Mediated Biodegradable Theranostic Nanoparticles for Melanoma Targeting and Drug Delivery

Although tremendous efforts have been made on targeted drug delivery systems, current therapy outcomes still suffer from low circulating time and limited targeting efficiency. The integration of cell‐mediated drug delivery and theranostic nanomedicine can potentially improve cancer management in both therapeutic and diagnostic applications. By taking advantage of innate immune cell's ability to target tumor cells, the authors develop a novel drug delivery system by using macrophages as both nanoparticle (NP) carriers and navigators to achieve cancer‐specific drug delivery. Theranostic NPs are fabricated from a unique polymer, biodegradable photoluminescent poly (lactic acid) (BPLP‐PLA), which possesses strong fluorescence, biodegradability, and cytocompatibility. In order to minimize the toxicity of cancer drugs to immune cells and other healthy cells, an anti‐BRAF V600E mutant melanoma specific drug (PLX4032) is loaded into BPLP‐PLA nanoparticles. Muramyl tripeptide is also conjugated onto the nanoparticles to improve the nanoparticle loading efficiency. The resulting nanoparticles are internalized within macrophages, which are tracked via the intrinsic fluorescence of BPLP‐PLA. Macrophages carrying nanoparticles deliver drugs to melanoma cells via cell–cell binding. Pharmacological studies also indicate that the PLX4032 loaded nanoparticles effectively kill melanoma cells. The “self‐powered” immune cell‐mediated drug delivery system demonstrates a potentially significant advancement in targeted theranostic cancer nanotechnologies.     

Single siRNA Nanocapsules for Effective siRNA Brain Delivery and Glioblastoma Treatment

Small interfering RNA (siRNA) has been considered as a highly promising therapeutic agent for human cancer treatment including glioblastoma (GBM), which is a fatal disease without effective therapy methods. However, siRNA-based GBM therapy is seriously hampered by a number of challenges in siRNA brain delivery including poor stability, short blood circulation, low blood–brain barrier (BBB) penetration, and tumor accumulation, as well as inefficient siRNA intracellular release. Herein, an Angiopep-2 (Ang) functionalized intracellular-environment-responsive siRNA nanocapsule (Ang-NC_ss(siRNA)) is successfully developed as a safe and efficient RNAi agent to boost siRNA-based GBM therapy. The experimental results demonstrate that the developed Ang-NC_ss(siRNA) displays long circulation in plasma, efficient BBB penetration capability, and GBM accumulation and retention, as well as responsive intracellular siRNA release due to the unique design of small size (25 nm) with polymeric shell for siRNA protection, Ang functionalization for BBB crossing and GBM targeting, and disulfide bond as a linker for intracellular-environment-responsive siRNA release. Such superior properties of Ang-NC_ss(siRNA) result in outstanding growth inhibition of orthotopic U87MG xenografts without causing adverse effects, achieving remarkably improved survival benefits. The developed siRNA nanocapsules provide a new strategy for RNAi therapy of GBM and beyond.     

Nanoparticles for Gene Delivery

Nanocarriers are a new type of nonviral gene carriers, many of which have demonstrated a broad range of pharmacological and biological properties, such as being biodegradable in the body, stimulus‐responsive towards the surrounding environment, and an abiltiy to specifically targeting certain disease sites. By summarizing some main types of nanocarriers, this Concept considers the current status and possible future directions of the potential clinical applications of multifunctional nanocarriers, with primary attention on the combination of such properties as biodegradability, targetability, transfection ability, and stimuli sensitivity.     

pH‐Responsive Isoniazid‐Loaded Nanoparticles Markedly Improve Tuberculosis Treatment in Mice

Tuberculosis is a major global health problem for which improved therapeutics are needed to shorten the course of treatment and combat emergence of drug resistance. Mycobacterium tuberculosis, the etiologic agent of tuberculosis, is an intracellular pathogen of mononuclear phagocytes. As such, it is an ideal pathogen for nanotherapeutics because macrophages avidly ingest nanoparticles even without specific targeting molecules. Hence, a nanoparticle drug delivery system has the potential to target and deliver high concentrations of drug directly into M. tuberculosis‐infected cells—greatly enhancing efficacy while avoiding off‐target toxicities. Stimulus‐responsive mesoporous silica nanoparticles of two different sizes, 100 and 50 nm, are developed as carriers for the major anti‐tuberculosis drug isoniazid in a prodrug configuration. The drug is captured by the aldehyde‐functionalized nanoparticle via hydrazone bond formation and coated with poly(ethylene imine)–poly(ethylene glycol) (PEI–PEG). The drug is released from the nanoparticles in response to acidic pH at levels that naturally occur within acidified endolysosomes. It is demonstrated that isoniazid‐loaded PEI–PEG‐coated nanoparticles are avidly ingested by M. tuberculosis‐infected human macrophages and kill the intracellular bacteria in a dose‐dependent manner. It is further demonstrated in a mouse model of pulmonary tuberculosis that the nanoparticles are well tolerated and much more efficacious than an equivalent amount of free drug.