Synthesis and characterization of stable fluorocarbon nanostructures as drug delivery vehicles for cytolytic peptides

The therapeutic potential of cytolytic peptides is plagued by nonspecificity and enzymatic degradation. We report the first stable incorporation of melittin (a 26 amino acid amphipathic peptide) into an outer lipid monolayer of perfluorocarbon nanoparticles. Melittin binds avidly to the nanoparticles (dissociation constant approximately 3.27 nM) and retains its pore-forming activity after contact-mediated delivery to model bilayer membrane (liposomes) thereby demonstrating the effectiveness of perfluorocarbon nanoparticles as unique nanocarriers for cytolytic peptides.     

Porous Silicon Nanoparticle Delivery of Tandem Peptide Anti‐Infectives for the Treatment of Pseudomonas aeruginosa Lung Infections

There is an urgent need for new materials to treat bacterial infections. In order to improve antibacterial delivery, an anti‐infective nanomaterial is developed that utilizes two strategies for localization: i) a biodegradable nanoparticle carrier to localize therapeutics within the tissue, and ii) a novel tandem peptide cargo to localize payload to bacterial membranes. First, a library of antibacterial peptides is screened that combines a membrane‐localizing peptide with a toxic peptide cargo and discovers a tandem peptide that displays synergy between the two domains and is able to kill Pseudomonas aeruginosa at sub‐micromolar concentrations. To apply this material to the lung, the tandem peptide is loaded into porous silicon nanoparticles (pSiNPs). Charged peptide payloads are loaded into the pores of the pSiNP at ≈30% mass loading and ≈90% loading efficiency using phosphonate surface chemistry. When delivered to the lungs of mice, this anti‐infective nanomaterial exhibits improved safety profiles over free peptides. Moreover, treatment of a lung infection of P. aeruginosa results in a large reduction in bacterial numbers and markedly improves survival compared to untreated mice. Collectively, this study presents the selection of a bifunctional peptide‐based anti‐infective agent and its delivery via biodegradable nanoparticles for application to an animal model of lung infection.     

Endosomal escape of protein nanoparticles engineered through humanized histidine-rich peptides

Poly-histidine peptides such as H6(HHHHHH)are used in protein biotechnologies as purification tags,protein-assembling agents and endosomal-escape entities.The pleiotropic properties of such peptides make them appealing to design protein-based smart materials or nanoparticles for imaging or drug delivery to be produced in form of recombinant proteins.However,the clinical applicability of H6-tagged proteins is restricted by the potential immunogenicity of these segments.In this study,we have explored several humanized histidine-rich peptides in tumor-targeted modular proteins,which can specifically bind and be internalized by the target cells through the tumoral marker CXCR4.We were particularly interested in exploring how protein purification,self-assembling and endosomal escape perform in proteins containing the variant histidine-rich tags.Among the tested candidates,the peptide H5 E(HEHEHEHEH)is promising as a good promoter of endosomal escape of the associated fulllength protein upon endosomal internalization.The numerical modelling of cell penetration and endosomal escape of the tested proteins has revealed a negative relationship between the amount of protein internalized into target cells and the efficiency of cytoplasmic release.This fact demonstrates that the His-mediated,proton sponge-based endosomal escape saturates at moderate amounts of internalized protein,a fact that might be critical for the design of protein materials for cytosolic molecular delivery.     

pH‐Responsive PEG‐Shedding and Targeting Peptide‐Modified Nanoparticles for Dual‐Delivery of Irinotecan and microRNA to Enhance Tumor‐Specific Therapy

Irinotecan is one of the main chemotherapeutic agents for colorectal cancer (CRC). MicroRNA‐200 (miR‐200) has been reported to inhibit metastasis in cancer cells. Herein, pH‐sensitive and peptide‐modified liposomes and solid lipid nanoparticles (SLN) are designed for encapsulation of irinotecan and miR‐200, respectively. These peptides include one cell‐penetrating peptide, one ligand targeted to tumor neovasculature undergoing angiogenesis, and one mitochondria‐targeting peptide. The peptide‐modified nanoparticles are further coated with a pH‐sensitive PEG‐lipid derivative with an imine bond. These specially‐designed nanoparticles exhibit pH‐responsive release, internalization, and intracellular distribution in acidic pH of colon cancer HCT116 cells. These nanoparticles display low toxicity to blood and noncancerous intestinal cells. Delivery of miR‐200 by SLN further increases the cytotoxicity of irinotecan‐loaded liposomes against CRC cells by triggering apoptosis and suppressing RAS/β‐catenin/ZEB/multiple drug resistance (MDR) pathways. Using CRC‐bearing mice, the in vivo results further indicate that irinotecan and miR‐200 in pH‐responsive targeting nanoparticles exhibit positive therapeutic outcomes by inhibiting colorectal tumor growth and reducing systemic toxicity. Overall, successful delivery of miR and chemotherapy by multifunctional nanoparticles may modulate β‐catenin/MDR/apoptosis/metastasis signaling pathways and induce programmed cancer cell death. Thus, these pH‐responsive targeting nanoparticles may provide a potential regimen for effective treatment of colorectal cancer.     

Site‐Specific Modification of Adeno‐Associated Viruses via a Genetically Engineered Aldehyde Tag

As a consequence of their well‐defined nanostructure and intrinsic bioactive functionality, virus‐based nanoparticles have shown promise for mediating gene delivery. Adeno‐associated virus (AAV) nanoparticles, which possess an excellent safety profile and therapeutic potential, hold potential for use in human gene therapy. However, because of their native tropisms, the applicability of AAV nanoparticles is often limited to restricted ranges of cells or tissues. Thus, retargeting AAV particles to the desired cell populations has continued to be a major research focus in many gene therapy applications. In this study, a general strategy is reported for nanoparticle targeting. This involves the site‐specific modification of AAV type 2 (AAV2) by genetically incorporating a short peptide, in this case an aldehyde tag, in the viral capsid. Such a tag can be exploited for site‐specific attachment of targeting molecules and allows for further introduction of targeting antibodies or ligands. It is shown that this modification neither affects the level of infectious viral titer nor intracellular trafficking properties. Furthermore, the site‐specific conjugation of targeting antibodies could significantly enhance viral transduction to those target cells that have otherwise exhibited very low permissiveness to AAV2 infection. This method also allows the functional incorporation of RGD peptides onto AAV2 for enhanced delivery with implications for cancer gene therapy.     

In vivo Gold Nanoparticle Delivery of Peptide Vaccine Induces Anti‐Tumor Immune Response in Prophylactic and Therapeutic Tumor Models

Gold nanoparticles (AuNPs) are promising vehicles for cancer immunotherapy, with demonstrated efficacy in immune delivery and innate cell stimulation. Nevertheless, their potential has yet to be assessed in the in vivo application of peptide cancer vaccines. In this study, it is hypothesized that the immune distribution and adjuvant qualities of AuNPs could be leveraged to facilitate delivery of the ovalbumin (OVA) peptide antigen and the CpG adjuvant and enhance their therapeutic effect in a B16‐OVA tumor model. AuNP delivery of OVA (AuNP‐OVA) and of CpG (AuNP‐CpG) enhanced the efficacy of both agents and induced strong antigen‐specific responses. In addition, it is found that AuNP‐OVA delivery alone, without CpG, is sufficient to promote significant antigen‐specific responses, leading to subsequent anti‐tumor activity and prolonged survival in both prophylactic and therapeutic in vivo tumor models. This enhanced therapeutic efficacy is likely due to the adjuvant effect of peptide coated AuNPs, as they induce inflammatory cytokine release when cultured with bone marrow dendritic cells. Overall, AuNP‐mediated OVA peptide delivery can produce significant therapeutic benefits without the need of adjuvant, indicating that AuNPs are effective peptide vaccine carriers with the potential to permit the use of lower and safer adjuvant doses during vaccination.     

Programmed Nanoparticle‐Loaded Nanoparticles for Deep‐Penetrating 3D Cancer Therapy

Tumors are 3D, composed of cellular agglomerations and blood vessels. Therapies involving nanoparticles utilize specific accumulations due to the leaky vascular structures. However, systemically injected nanoparticles are mostly uptaken by cells located on the surfaces of cancer tissues, lacking deep penetration into the core cancer regions. Herein, an unprecedented strategy, described as injecting “nanoparticle‐loaded nanoparticles” to address the long‐lasting problem is reported for effective surface‐to‐core drug delivery in entire 3D tumors. The “nanoparticle‐loaded nanoparticle” is a silica nanoparticle (≈150 nm) with well‐developed, interconnected channels (diameter of ≈30 nm), in which small gold nanoparticles (AuNPs) (≈15 nm) with programmable DNA are located. The nanoparticle (AuNPs)‐loaded nanoparticles (silica): (1) can accumulate in tumors through leaky vascular structures by protecting the inner therapeutic AuNPs during blood circulation, and then (2) allow diffusion of the AuNPs for penetration into the entire surface‐to‐core tumor tissues, and finally (3) release a drug triggered by cancer‐characteristic pH gradients. The hierarchical “nanoparticle‐loaded nanoparticle” can be a rational design for cancer therapies because the outer large nanoparticles are effective in blood circulation and in protection of the therapeutic nanoparticles inside, allowing the loaded small nanoparticles to penetrate deeply into 3D tumors with anticancer drugs.     

Development of Novel Tumor‐Targeted Theranostic Nanoparticles Activated by Membrane‐Type Matrix Metalloproteinases for Combined Cancer Magnetic Resonance Imaging and Therapy

A major drawback with current cancer therapy is the prevalence of unrequired dose‐limiting toxicity to non‐cancerous tissues and organs, which is further compounded by a limited ability to rapidly and easily monitor drug delivery, pharmacodynamics and therapeutic response. In this report, the design and characterization of novel multifunctional “theranostic” nanoparticles (TNPs) is described for enzyme‐specific drug activation at tumor sites and simultaneous in vivo magnetic resonance imaging (MRI) of drug delivery. TNPs are synthesized by conjugation of FDA‐approved iron oxide nanoparticles ferumoxytol to an MMP‐activatable peptide conjugate of azademethylcolchicine (ICT), creating CLIO‐ICTs (TNPs). Significant cell death is observed in TNP‐treated MMP‐14 positive MMTV‐PyMT breast cancer cells in vitro, but not MMP‐14 negative fibroblasts or cells treated with ferumoxytol alone. Intravenous administration of TNPs to MMTV‐PyMT tumor‐bearing mice and subsequent MRI demonstrates significant tumor selective accumulation of the TNP, an observation confirmed by histopathology. Treatment with CLIO‐ICTs induces a significant antitumor effect and tumor necrosis, a response not observed with ferumoxytol. Furthermore, no toxicity or cell death is observed in normal tissues following treatment with CLIO‐ICTs, ICT, or ferumoxytol. These findings demonstrate proof of concept for a new nanotemplate that integrates tumor specificity, drug delivery and in vivo imaging into a single TNP entity through attachment of enzyme‐activated prodrugs onto magnetic nanoparticles. This novel approach holds the potential to significantly improve targeted cancer therapies, and ultimately enable personalized therapy regimens.     

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.     

Nanoparticle‐Induced Folding and Fibril Formation of Coiled‐Coil‐Based Model Peptides

Nanomedicine is a rapidly growing field that has the potential to deliver treatments for many illnesses. However, relatively little is known about the biological risks of nanoparticles. Some studies have shown that nanoparticles can have an impact on the aggregation properties of proteins, including fibril formation. Moreover, these studies also show that the capacity of nanoscale objects to induce or prevent misfolding of the proteins strongly depends on the primary structure of the protein. Herein, light is shed on the role of the peptide primary structure in directing nanoparticle‐induced misfolding by means of two model peptides. The design of these peptides is based on the α‐helical coiled‐coil folding motif, but also includes features that enable them to respond to pH changes, thus allowing pH‐dependent β‐sheet formation. Previous studies showed that the two peptides differ in the pH range required for β‐sheet folding. Time‐dependent circular dichroism spectroscopy and transmission electron microscopy are used to characterize peptide folding and aggregate morphology in the presence of negatively charged gold nanoparticles (AuNPs). Both peptides are found to undergo nanoparticle‐induced fibril formation. The determination of binding parameters by isothermal titration calorimetry further reveals that the different propensities of both peptides to form amyloid‐like structures in the presence of AuNPs is primarily due to the binding stoichiometry to the AuNPs. Modification of one of the peptide sequences shows that AuNP‐induced β‐sheet formation is related to the structural propensity of the primary structure and is not a generic feature of peptide sequences with a sufficiently high binding stoichiometry to the nanoparticles.     

Advances in Peptide Functionalization on Mesoporous Silica Nanoparticles for Controlled Drug Release

During the last decade, using versatile, promising, and fascinating mesoporous silica nanoparticles (MSNs) as site‐specific and stimuli‐responsive drug delivery systems (DDSs) has received concentrated research interest. As one of the most attractive surface modification units, peptides have inherent bioactivity, biodegradability and biocompatibility. Recent progresses in the utilization of versatile peptides for surface functionalization of MSNs to achieve cell‐specific targeting, fluorescence imaging, and intracellular diagnosis and treatment of tumors are summarized in this review. The various functional peptides decorated on the MSNs are introduced and classified into three types, including targeting peptides, stimuli‐responsive peptides and multifunctional chimeric peptides. The limitations and challenges of peptide modified MSNs and their potential applications are further discussed.     

Peptide‐Based Nanotubes and Their Applications in Bionanotechnology

In nature, biological nanomaterials are synthesized under ambient conditions in a natural microscopic‐sized laboratory, such as a cell. Biological molecules, such as peptides and proteins, undergo self‐assembly processes in vivo and in vitro, and these monomers are assembled into various nanometer‐scale structures at room temperature and atmospheric pressure. The self‐assembled peptide nanostructures can be further organized to form nanowires, nanotubes, and nanoparticles via their molecular‐recognition functions. The application of molecular self‐assemblies of synthetic peptides as nanometer‐scale building blocks in devices is robust, practical, and affordable due to their advantages of reproducibility, large‐scale production ability, monodispersity, and simpler experimental methods. It is also beneficial that smart functionalities can be added at desired positions in peptide nanotubes through well‐established chemical and peptide syntheses. These features of peptide‐based nanotubes are the driving force for investigating and developing peptide nanotube assemblies for biological and non‐biological applications.     

Fluorinated Polymer Mediated Transmucosal Peptide Delivery for Intravesical Instillation Therapy of Bladder Cancer

Surgical intervention combined with intravesical instillation of chemotherapeutics to clear residual cancer cells after operation is the current standard treatment method for bladder cancer. However, the poor bioavailability of active pharmaceutical ingredients for bladder cancer cells on account of the biological barriers of bladder mucosa, together with significant side effects of currently used intravesical medicine, have limited the clinical outcomes of localized adjuvant therapy for bladder cancer. Aiming at improved intravesical instillation therapy of bladder cancer, a fluorinated polyethylenimine (F‐PEI) is employed here for the transmucosal delivery of an active venom peptide, polybia‐mastoparan I (MPI), which shows selective antiproliferative effect against various bladder cancer cell lines. Upon simple mixing, MPI and F‐PET would coassemble to form stable nanoparticles, which show greatly improved cross‐membrane and transmucosal penetration capacities compared with MPI alone or nonfluorinated MPI/PEI nanoparticles. MPI/F‐PEI shows higher in vivo tumor growth inhibition efficacy for local treatment of a subcutaneous tumor model. More excitingly, as further demonstrated in an orthotopic bladder cancer model, MPI/F‐PEI offers remarkably improved therapeutic effects compared to those achieved by free MPI or the first‐line bladder cancer drug mitomycin C. This work presents a new transmucosal delivery carrier particularly promising for intravesical instillation therapy of bladder cancer.     

Probing Cell‐Type‐Specific Intracellular Nanoscale Barriers Using Size‐Tuned Quantum Dots

The compartmentalization of size‐tuned luminescent semiconductor nanocrystal quantum dots (QDs) in four distinctive cell lines, which would be representative of the most likely environmental exposure routes to nanoparticles in humans, is studied. The cells are fixed and permeabilized prior to the addition of the QDs, thus eliminating any cell‐membrane‐associated effects due to active QD uptake mechanisms or to specificity of signaling routes in different cell types, but leaving intact the putative physical subcellular barriers. All quantitative assays are performed using a high content analysis (HCA) platform, thereby obtaining robust data on large cell populations. While smaller QDs 2.1 nm in diameter enter the nuclei and localize to the nucleoli in all cell types, the rate and dynamics of their passage vary depending on the cell origin. As the QD size is increased to 4.4 nm, penetration into the cell is reduced but each cell line displays its own cutoff size thresholds reflecting cell‐type‐determined cytoplasmic and nuclear pore penetration specificity. These results give rise to important considerations regarding the differential compartmentalization and susceptibility of organs, tissues, and cells to nanoparticles, and may be of prime importance for biomedical imaging and drug‐delivery research employing nanoparticle‐based probes and systems.     

Enzymatic activation of a peptide functionalised gold nanoparticle system for prodrug delivery

Gold nanoparticles (GNPs) with a monolayer of peptides were synthesized as a potential tumour activated cancer drug delivery system. The prodrug system was achieved by the attachment of two varying lengths of peptides to GNPs: An 18 amino acid peptide sequence encompassing a shorter fluorescent labelled (coumarin) six amino acid peptide sequence. The longer peptide chain included the sequence D-AFK that is selectively cleavable by the over-expression of proteases in the vicinity of cancer cells. The protease-mediated exposure of the coumarin was demonstrated by the incubation of peptide capped GNPs with adenocarcinomic human alveolar basal epithelial A549 cells and madin-darby bovine kidney epithelial cells. Confocal laser scanning microscopy studies revealed enhanced fluorescence emission intensities in the cancer cell line as compared to the intensity exhibited by the healthy cell line. This work suggests that GNPs functionalised with a cytotoxic agent or fluorophore encapsulated by longer peptide strands may find useful applications for development of GNPs with therapeutic or diagnostic studies.     

Layer‐by‐Layer Coated Gold Nanoparticles: Size‐Dependent Delivery of DNA into Cells

Because nanoparticles are finding uses in myriad biomedical applications, including the delivery of nucleic acids, a detailed knowledge of their interaction with the biological system is of utmost importance. Here the size‐dependent uptake of gold nanoparticles (AuNPs) (20, 30, 50 and 80 nm), coated with a layer‐by‐layer approach with nucleic acid and poly(ethylene imine) (PEI), into a variety of mammalian cell lines is studied. In contrast to other studies, the optimal particle diameter for cellular uptake is determined but also the number of therapeutic cargo molecules per cell. It is found that 20 nm AuNPs, with diameters of about 32 nm after the coating process and about 88 nm including the protein corona after incubation in cell culture medium, yield the highest number of nanoparticles and therapeutic DNA molecules per cell. Interestingly, PEI, which is known for its toxicity, can be applied at significantly higher concentrations than its IC₅₀ value, most likely because it is tightly bound to the AuNP surface and/or covered by a protein corona. These results are important for the future design of nanomaterials for the delivery of nucleic acids in two ways. They demonstrate that changes in the nanoparticle size can lead to significant differences in the number of therapeutic molecules delivered per cell, and they reveal that the toxicity of polyelectrolytes can be modulated by an appropriate binding to the nanoparticle surface.     

ROS‐Responsive Polyprodrug Nanoparticles for Triggered Drug Delivery and Effective Cancer Therapy

The application of nanoparticles (NPs) to drug delivery has led to the development of novel nanotherapeutics for the treatment of various diseases including cancer. However, clinical use of NP‐mediated drug delivery has not always translated into improved survival of cancer patients, in part due to the suboptimal properties of NP platforms, such as premature drug leakage during preparation, storage, or blood circulation, lack of active targeting to tumor tissue and cells, and poor tissue penetration. Herein, an innovative reactive oxygen species (ROS)‐responsive polyprodrug is reported that can self‐assemble into stable NPs with high drug loading. This new NP platform is composed of the following key components: (i) polyprodrug inner core that can respond to ROS for triggered release of intact therapeutic molecules, (ii) polyethylene glycol (PEG) outer shell to prolong blood circulation; and (iii) surface‐encoded internalizing RGD (iRGD) to enhance tumor targeting and tissue penetration. These targeted ROS‐responsive polyprodrug NPs show significant inhibition of tumor cell growth both in vitro and in vivo.     

Polyhedral Oligomeric Silsesquioxane-Based Nanoparticles for Efficient Chemotherapy of Glioblastoma

Glioblastoma (GBM) is the most common lethal brain tumor with dismal treatment outcomes and poor response to chemotherapy. As the regulatory center of cytogenetics and metabolism, most tumor chemotherapeutic molecules exert therapeutic effects in the nucleus. Nanodrugs showing the nuclear aggregation effect are expected to eliminate and fundamentally suppress tumor cells. In this study, a nanodrug delivery system based on polyhedral oligomeric silsesquioxane (POSS) is introduced to deliver drugs into the nuclei of GBM cells, effectively enhancing the therapeutic efficacy of chemotherapy. The nanoparticles are modified with folic acid and iRGD peptides molecules to improve their tumor cell targeting and uptake via receptor-mediated endocytosis. Nuclear aggregation allows for the direct delivery of chemotherapeutic drug temozolomide (TMZ) to the tumor cell nuclei, resulting in more significant DNA damage and inhibition of tumor cell proliferation. Herein, TMZ-loaded POSS nanoparticles can significantly improve the survival of GBM-bearing mice. Therefore, the modified POSS nanoparticles may serve as a promising drug-loaded delivery platform to improve chemotherapy outcomes in GBM patients.     

Hierarchical Targeting Strategy for Enhanced Tumor Tissue Accumulation/Retention and Cellular Internalization

Targeted delivery of therapeutic agents is an important way to improve the therapeutic index and reduce side effects. To design nanoparticles for targeted delivery, both enhanced tumor tissue accumulation/retention and enhanced cellular internalization should be considered simultaneously. So far, there have been very few nanoparticles with immutable structures that can achieve this goal efficiently. Hierarchical targeting, a novel targeting strategy based on stimuli responsiveness, shows good potential to enhance both tumor tissue accumulation/retention and cellular internalization. Here, the recent design and development of hierarchical targeting nanoplatforms, based on changeable particle sizes, switchable surface charges and activatable surface ligands, will be introduced. In general, the targeting moieties in these nanoplatforms are not activated during blood circulation for efficient tumor tissue accumulation, but re‐activated by certain internal or external stimuli in the tumor microenvironment for enhanced cellular internalization.