Exploring the outer limits of polyplexes.

Histidine-containing polymers show promise in their transport of nucleic acids in vitro and in vivo. In addition to the pH-buffering histidine component, the polymer often contains a protonated component at physiological pH, such as lysine. These polyplexes usually accumulate in the tumor by enhanced permeability and retention, which has proved disappointing in clinical trials. We presently compare two histidine-lysine (HK) peptide polyplexes for their neuropilin-1-mediated transport of plasmids in vivo. While the polymerized HK (H2KC-48) polyplex was markedly better than the monomeric HK (H2K) polyplex in vitro, both HK polyplexes were effective in transfecting tumor xenografts over a wide range of peptide and plasmid ratios. Nevertheless, polyplexes of low peptide/DNA ratios gave higher tumor transfection and specificity than those of higher ratios. Surprisingly, there was minimal to no gel retardation of polyplexes made from these low ratios during electrophoresis. These results demonstrate that loosely packed HK polyplexes effectively transfected tumors in vivo.

Marked increase in tumor transfection with a truncated branched polymer.

BACKGROUND: We previously determined that polyplexes formed by linear H2K peptides were more effective in transfecting tumors in vivo than polyplexes formed by branched H2K4b-20 peptides. Based on trypsin digest and salt displacement studies, the linear H2K polyplexes were less stable than the branched H2K4b-20 polyplexes. Because binding and release of the polymer and DNA from the H2K4b-20 polyplex may account for the ineffectiveness, we investigated whether four-branched histidine-lysine (HK) peptides with varying numbers of amino acids in their branches would be more effective in their ability to increase gene expression in tumors in vivo. METHODS: Linear and branched peptides with multiple -KHHK- motifs were synthesized by solid-phase synthesis. The branched H2K4b-20, -18, -14 and 12 peptides had 20, 18, 14 and 12 amino acids in their branches, respectively. These peptides were examined for their ability to carry luciferase-expressing plasmids to human breast cancer xenografts in a mouse model. With gel retardation and in vivo transfection, the incorporation of a targeting ligand and an endosomal lysis peptide into these polyplexes was also examined. A blocking antibody was pre-injected prior to the polyplexes to determine the role of neuropilin 1 in the uptake of these polyplexes by the tumor. The size of the polyplexes was measured by dynamic light scattering. RESULTS: Of the four negative surface-charge polyplexes formed by the branched carriers, the H2K4b-14 polyplex was determined to be the most effective plasmid delivery platform to tumors. The incorporation of a targeting ligand and an endosomal lysis peptide into H2K4b-14 polyplexes further enhanced their ability to transfect tumors in vivo. Furthermore, after pre-injecting tumor-bearing mice with a blocking antibody to the neuropilin-1 receptor (NRP-1), there was a marked reduction of tumor gene expression with the modified H2K4b-14 polyplexes, suggesting that NRP-1 mediated their transport into the tumor. CONCLUSIONS: The present study established that branched peptides intermediate in length were very efficient in delivering plasmids to tumors in vivo.

Location of a single histidine within peptide carriers increases mRNA delivery.

BACKGROUND: Previously, we determined that four-branched histidine-lysine (HK) peptides were effective carriers of plasmids and small interfering RNA. In the present study, we compared several branched HK carriers and, in particular, two closely-related H3K4b and H3K(+H)4b peptides for their ability as carriers of mRNA. The H3K(+H)4b peptide differed from its parent analogue, H3K4b, by only a single histidine in each branch. METHODS: A series of four-branched HK peptides with varied sequences was synthesized on a solid-phase peptide synthesizer. The ability of these peptides to carry mRNA expressing luciferase to MDA-MB-231 cells was investigated. With gel retardation and heparin displacement assays, the stability of HK polyplexes was examined. We determined the intracellular uptake of HK polyplexes by flow cytometry and fluorescence microscopy. The size and polydispersity index of the polyplexes in several media were measured by dynamic light scattering. RESULTS: MDA-MB-231 cells transfected by H3K(+H)4b-mRNA polyplexes expressed 10-fold greater levels of luciferase than H3K4b polyplexes. With gel retardation and heparin displacement assays, the H3K(+H)4b polyplexes showed greater stability than H3K4b. Intracellular uptake and co-localization of H3K(+H)4b polyplexes within acidic endosomes were also significantly increased compared to H3K4b. Similar to H3K(+H)4b, several HK analogues with an additional histidine in the second domain of their branches were effective carriers of mRNA. When combined with DOTAP liposomes, H3K(+H)4b was synergistic in delivery of mRNA. CONCLUSIONS: H3K(+H)4b was a more effective carrier of mRNA than H3K4b. Mechanistic studies suggest that H3K(+H)4b polyplexes were more stable than H3K4b polyplexes. Lipopolyplexes formed with H3K(+H)4b markedly increased mRNA transfection.

Enhanced tumor uptake and activity of nanoplex-loaded doxorubicin.

Doxorubicin (Dox) has widespread use as a cancer chemotherapeutic agent, but Dox is limited by several side effects including irreversible cardiomyopathy. Although liposomal Dox formulations, such as Doxil, mitigate side effects, they do not prolong survival in many patients. As a result, efforts have continued to discover improved formulations of Dox. We previously found that a peptide-based nanoplex delivered plasmid DNA efficiently to tumors in murine models. Unlike the majority of nanoparticles that depend solely on enhanced permeability and retention (EPR) for their transport into the tumor, our peptide-based nanoplex has a potential advantage in that its uptake primarily depends on neuropilin-1 receptor targeting. Because Dox binds to DNA, we tested whether this delivery platform could effectively deliver Dox to tumors and reduce their size. The nanoplexes increased the levels of Dox in tumors by about 5.5-fold compared to aqueous (free) Dox controls. Consistent with enhanced levels in the tumor, the nanoplex-Dox treatment had significantly greater anti-tumor activity. Whereas low dose free Dox did not reduce the size of tumors compared to untreated controls, the low dose nanoplex-Dox reduced the size of tumors by nearly 55% (p < 0.001). The high dose nanoplex-Dox also inhibited the size of tumor significantly more than the comparable high-dose free Dox (p < 0.001). Furthermore, apoptosis and proliferation markers (Ki67) of tumors observed in the different treatment groups correlated with their ability to inhibit tumor size. This study shows the efficacy of an NRP-1 targeted nanoplexes to deliver Dox to tumors in vivo and lays the groundwork for more complex and effective formulations.

NRP1 transport of cancer therapeutics mediated by tumor-penetrating peptides.

Whereas uptake of low molecular weight agents is generally inhibited in tumors due to high interstitial pressure, tumor uptake of macromolecules is increased due to enhanced permeability and retention (EPR). Small molecule drugs alone or incorporated in nanoparticles (NP) have largely been dependent on such physical tumor uptake (passive) for therapeutic activity. Although passive targeted NP such as Stealth Liposomal Doxorubicin (Doxil ®) are effective with improved safety, drug delivery to tumors is still significantly limited. To improve tumor delivery and efficacy, tumor-penetrating peptides (TPP), which contain sequences that target the tumor and activate the neuropilin-1 receptor (NRP1), have either been co-administered with or conjugated to both small and large therapeutic molecules. In this review, we will discuss TPP-mediated therapeutics which target the NRP1 transport system of tumors.

The neuropilin-1 receptor mediates enhanced tumor delivery of H2K polyplexes.

BACKGROUND: Promising plasmid-based treatments have limited value without an effective delivery system. Recently, the linear H2K with a repeating -KHHK- pattern was determined to be an effective plasmid carrier to tumor xenografts in vivo. Although unpacking of the H2K polyplex within the tumor may have a role, the mechanism for the enhanced efficacy remains unclear. METHODS: After solid-phase synthesis of linear and branched histidine-lysine (HK) peptide carriers of plasmids, the peptides were compared for their ability to lyse endosomes with a red blood cell model and to transfect MDA-MB-435 xenografts in the presence or absence of neuropilin-1 receptor (NRP-1) antibodies. To examine stability, polyplexes were incubated with trypsin or NaCl and then analyzed by electrophoresis. RESULTS: After screening peptides with a model for endosomal lysis at two pHs, the 33-mer H3K peptide lysed red blood cells effectively at the lower pH. Combining H3K and H2K peptides as carriers of plasmids expressing luciferase were more effective than H2K alone. Based on the repeating -KHHK- sequences of H2K, we studied whether the widespread gene expression in the tumor may be mediated by NRP-1. By blocking NRP-1 in tumor-bearing mice, luciferase activity in tumors delivered by HK polyplexes was reduced by 96%, whereas activity in normal tissues was minimally reduced. CONCLUSIONS: Combining an endosomolytic peptide, H3K, with H2K polyplexes as a carrier further enhanced transfection in vivo. Moreover, the widespread distribution of H2K polyplexes is mediated by NRP-1, suggesting that transcytosis of these polyplexes through the tumor endothelium may lead to efficient transfection. Copyright © 2016 John Wiley & Sons, Ltd.

Silver adducts of four-branched histidine rich peptides exhibit synergistic antifungal activity.

Previously, a four branched histidine-lysine rich peptide, H3K4b, was shown to demonstrate selective antifungal activity with minimal antibacterial activity. Due to the potential breakdown from proteases, H3K4b was further evaluated in the current study by varying the D- and l-amino acid content in its branches. Whereas analogues of H3K4b that selectively replaced l-amino acids (H3k4b, h3K4b) had improved antifungal activity, the all d-amino acid analogue, h3k4b, had reduced activity, suggesting that partial breakdown of the peptide may be necessary. Moreover, because histidines form coordination bonds with the silver ion, we examined whether silver adducts can be formed with these branched histidine-lysine peptides, which may improve antifungal activity. For Candida albicans, the silver adduct of h3K4b or H3k4b reduced the MIC compared to peptide and silver ions alone by 4- and 5-fold, respectively. For Aspergillus fumigatus, the silver adducts showed even greater enhancement of activity. Although the silver adducts of H3k4b or h3K4b showed synergistic activity, the silver adduct with the all l-amino acid H3K4b surprisingly showed the greatest synergistic and growth inhibition of A. fumigatus: the silver adduct of H3K4b reduced the MIC compared to the peptide and silver ions alone by 30- and 26-fold, respectively. Consistent with these antifungal efficacy results, marked increases in free oxygen radicals were produced with the H3K4b and silver combination. These studies suggest that there is a balance between stability and breakdown for optimal antifungal activity of the peptide alone and for the peptide-silver adduct.

Increased tumor distribution and expression of histidine-rich plasmid polyplexes.

BACKGROUND: Selecting nonviral carriers for in vivo gene delivery is often dependent on determining the optimal carriers from transfection assays in vitro. The rationale behind this in vitro strategy is to cast a net sufficiently wide to identify the few effective carriers of plasmids for in vivo studies. Nevertheless, many effective in vivo carriers may be overlooked by this strategy because of the marked differences between in vitro and in vivo assays. METHODS: After solid-phase synthesis of linear and branched histidine/lysine (HK) peptides, the two peptide carriers were compared for their ability to transfect MDA-MB-435 tumor cells in vitro and then in vivo. RESULTS: By contrast to their transfection activity in vitro, the linear H2K carrier of plasmids was far more effective in vivo compared to the branch H2K4b. Surprisingly, negatively-charged polyplexes formed by the linear H2K peptide gave higher transfection in vivo than did those with a positive surface charge. To examine the distribution of plasmid expression within the tumor from H2K polyplexes, we found widespread expression by immunohistochemical staining. With a fluorescent tdTomato expressing-plasmid, we confirmed a pervasive distribution and gene expression within the tumor mediated by the H2K polyplex. CONCLUSIONS: Although mechanisms underlying the efficiency of gene expression are probably multifactorial, unpacking of the H2K polyplex within the tumor appears to have a significant role. Further development of these H2K polyplexes represents an attractive approach for plasmid-based therapies of cancer.

Direct observation of dynamic mechanical regulation of DNA condensation by environmental stimuli.

Gene delivery is a promising way to treat hereditary diseases and cancer; however, there is little understanding of DNA:carrier complex mechanical properties, which may be critical for the protection and release of nucleic acids. We applied optical tweezers to directly measure single-molecule mechanical properties of DNA condensed using 19-mer poly-L-lysine (PLL) or branched histidine-lysine (HK) peptides. Force-extension profiles indicate that both carriers condense DNA actively, showing force plateaus during stretching and relaxation cycles. As the environment such as carrier concentration, pH, and the presence of zinc ions changes, DNA:HK complexes showed dynamically regulated mechanical properties at multiple force levels. The fundamental knowledge from this study can be applied to design a mechanically tailored complex which may enhance transfection efficiency by controlling the stability of the complex temporally and spatially.

Delivery of mRNA with Histidine-Lysine Peptides.

Transfection with mRNA has been considered superior to that with plasmids since the mRNA can be translated to a protein in the cytosol without entering the nucleus. One disadvantage of using mRNA is its susceptibility to enzymatic biodegradability, and consequently, significant research has occurred to determine nonviral carriers that will sufficiently stabilize this nucleic acid for cellular transport. Histidine-lysine peptides (HK) are one such class of mRNA carriers, which we think serves as a model for other peptides and polymeric carrier systems. When the HK peptide and mRNA are mixed and interact through ionic and nonionic bonds, mRNA polyplexes are formed, which can transfect cells. In contrast to linear HK peptides, branched HK peptides protected and efficiently transfected mRNA into cells. After describing the preparation and biophysical characterization of these polyplexes, we will provide protocols for in vitro and in vivo transfection for these mRNA polyplexes.

Surface-modified HK:siRNA nanoplexes with enhanced pharmacokinetics and tumor growth inhibition.

We characterized in this study the pharmacokinetics and antitumor efficacy of histidine-lysine (HK):siRNA nanoplexes modified with PEG and a cyclic RGD (cRGD) ligand targeting αvß3 and αvß5 integrins. With noninvasive imaging, systemically administered surface-modified HK:siRNA nanoplexes showed nearly 4-fold greater blood levels, 40% higher accumulation in tumor tissue, and 60% lower luciferase activity than unmodified HK:siRNA nanoplexes. We then determined whether the surface-modified HK:siRNA nanoplex carrier was more effective in reducing MDA-MB-435 tumor growth with an siRNA targeting Raf-1. Repeated systemic administration of the selected surface modified HK:siRNA nanoplexes targeting Raf-1 showed 35% greater inhibition of tumor growth than unmodified HK:siRNA nanoplexes and 60% greater inhibition of tumor growth than untreated mice. The improved blood pharmacokinetic results and tumor localization observed with the integrin-targeting surface modification of HK:siRNA nanoplexes correlated with greater tumor growth inhibition. This investigation reveals that through control of targeting ligand surface display in association with a steric PEG layer, modified HK: siRNA nanoplexes show promise to advance RNAi therapeutics in oncology and potentially other critical diseases.

Buffering capacity and size of siRNA polyplexes influence cytokine levels.

Induction of cytokines by small interfering RNA (siRNA) polyplexes has been a significant concern of researchers attempting to minimize the toxicity of this promising therapy. Although cationic carriers of siRNA are known to increase cytokine levels, few systematic studies have been done to determine what properties of the carrier are important to modulate cytokines. Because branched histidine-lysine (HK) peptides are effective carriers of siRNA and their sequence can be readily modified, we selected this class of carrier to determine which sequences of the peptide were important for cytokine induction. With the use of peripheral blood mononuclear cells (PBMCs), the HK peptide with a higher number of histidines (H3K(+H)4b) in complex with siRNA induced lower levels of cytokines compared with other HK (e.g., H2K4b, H3K4b, H3K(+N)4b) siRNA nanoplexes. Notably, these peptides' siRNA polyplexes showed a similar pattern of cytokine induction when injected intravenously in a mouse model, i.e., the HK with higher content of histidines induced cytokines the least. As indicated by the pH-sensitive dye within acidic endosomes, the greater pH-buffering capacity of H3K(+H)4b compared with other HK peptides may explain why cytokine levels were reduced. In addition to buffering capacity, the size of HK polyplexes markedly influenced cytokine production.

Delivery of Chemotherapy Agents and Nucleic Acids with pH-Dependent Nanoparticles.

With less than one percent of systemically injected nanoparticles accumulating in tumors, several novel approaches have been spurred to direct and release the therapy in or near tumors. One such approach depends on the acidic pH of the extracellular matrix and endosomes of the tumor. With an average pH of 6.8, the extracellular tumor matrix provides a gradient for pH-responsive particles to accumulate, enabling greater specificity. Upon uptake by tumor cells, nanoparticles are further exposed to lower pHs, reaching a pH of 5 in late endosomes. Based on these two acidic environments in the tumor, various pH-dependent targeting strategies have been employed to release chemotherapy or the combination of chemotherapy and nucleic acids from macromolecules such as the keratin protein or polymeric nanoparticles. We will review these release strategies, including pH-sensitive linkages between the carrier and hydrophobic chemotherapy agent, the protonation and disruption of polymeric nanoparticles, an amalgam of these first two approaches, and the release of polymers shielding drug-loaded nanoparticles. While several pH-sensitive strategies have demonstrated marked antitumor efficacy in preclinical trials, many studies are early in their development with several obstacles that may limit their clinical use.

Novel Method for Rapid Detection of COVID-19 Omicron Variant and Future Emerging Variants.

Over six million people have died worldwide as a result of SARS-CoV-19 (Covid-19). Several major and minor variant waves of Covid-19 including the Alpha (B.1.1.7), Delta (B.1.617.2), and the Omicron (B.1.1.529) have infected patients in the last two years. These individual variants have been associated with different infectivity and death rates, and it is anticipated that other variants of significance will occur. As a result, the emergence and frequency of the different Covid-19 variants at various locations in the world are critically important to monitor. To date, sequencing of these variants has been the primary method to track the prevalence of the different variants, but this approach is relatively slow and expensive. In contrast, we have developed two qPCR methods that are sensitive, specific, and economical which can distinguish the Omicron variant from the other variants. The first qPCR method detects a specific base mutation in Omicron enabling improved amplification. The second method is based on designing a primer specific for the region in which a deletion and an insertion have occurred in the Omicron variant. While the first approach can be readily adapted to identify other variants with point mutations, the second method can identify emerging deletion/insertion mutations such as BA.2, a subvariant of the Omicron.

pH-Sensitive Targeting of Tumors with Chemotherapy-Laden Nanoparticles: Progress and Challenges.

Accumulating chemotherapeutic drugs such as doxorubicin within a tumor while limiting the drug dose to normal tissues is a central goal of drug delivery with nanoparticles. Liposomal products such as Doxil® represent one of the marked successes of nanoparticle-based strategies. To replicate this success for cancer treatment, many approaches with nanoparticles are being explored in order to direct and release chemotherapeutic agents to achieve higher accumulation in tumors. A promising approach has been stimulus-based therapy, such as the release of chemotherapeutic agents from the nanoparticles in the acidic environments of the tumor matrix or the tumor endosomes. Upon reaching the acidic environments of the tumor, the particles, which are made up of pH-dependent polymers, become charged and release the entrapped chemotherapy agents. This review discusses recent advances in and prospects for pH-dependent histidine-based nanoparticles that deliver chemotherapeutic agents to tumors. The strategies used by investigators include an array of histidine-containing peptides and polymers which form micelles, mixed micelles, nanovesicles, polyplexes, and coat particles. To date, several promising histidine-based nanoparticles have been demonstrated to produce marked inhibition of tumor growth, but challenges remain for successful outcomes in clinical trials. The lessons learned from these histidine-containing particles will provide insight in the development of improved pH-dependent polymeric delivery systems for chemotherapy.

The Multifaceted Histidine-Based Carriers for Nucleic Acid Delivery: Advances and Challenges.

Histidines incorporated into carriers of nucleic acids may enhance the extracellular stability of the nanoparticle, yet aid in the intracellular disruption of the nanoparticle, enabling the release of the nucleic acid. Moreover, protonation of histidines in the endosomes may result in endosomal swelling with subsequent lysis. These properties of histidine are based on its five-member imidazole ring in which the two nitrogen atoms may form hydrogen bonds or act as a base in acidic environments. A wide variety of carriers have integrated histidines or histidine-rich domains, which include peptides, polyethylenimine, polysaccharides, platform delivery systems, viral phages, mesoporous silica particles, and liposomes. Histidine-rich carriers have played key roles in our understanding of the stability of nanocarriers and the escape of the nucleic acids from endosomes. These carriers show great promise and offer marked potential in delivering plasmids, siRNA, and mRNA to their intracellular targets.

Advances in Systemic siRNA Delivery.

Sequence-specific gene silencing with small interfering RNA (siRNA) has transformed basic science research, and the efficacy of siRNA therapeutics toward a variety of diseases is now being evaluated in pre-clinical and clinical trials. Despite its potential value, the highly negatively charged siRNA has the classic delivery problem of requiring transport across cell membranes to the cytosol. Consequently, carrier development for siRNA delivery is one of the most important problems to solve before siRNA can achieve widespread clinical use. An assortment of non-viral carriers including liposomes, peptides, polymers, and aptamers are being evaluated for their ability to shepherd siRNA to the target tissue and cross the plasma membrane barrier into the cell. Several promising carriers with low toxicity and increased specificity for disease targets have emerged for siRNA-based therapeutics. This review will discuss non-viral approaches for siRNA therapeutics, with particular focus on synthetic carriers for in vivo systemic delivery of siRNA.

Human rhomboid family-1 gene silencing causes apoptosis or autophagy to epithelial cancer cells and inhibits xenograft tumor growth.

The rhomboid family of genes carry out a wide range of important functions in a variety of organisms. Little is known, however, about the function of the human rhomboid family-1 gene (RHBDF1). We show here that RHBDF1 function is essential to epithelial cancer cell growth. RHBDF1 mRNA level is significantly elevated in clinical specimens of invasive ductal carcinoma of the breast, and the protein is readily detectable in human breast cancer or head and neck cancer cell lines. Silencing the RHBDF1 gene with short interfering RNA (siRNA) results in apoptosis in breast cancer MDA-MB-435 cells and autophagy in head and neck squamous cell cancer 1483 cells. The treatment also leads to significant down-modulation of activated AKT and extracellular signal-regulated kinase in the cells, suggesting that critically diminished strength of these growth signals may be the key attributes of the induction of cell death. Furthermore, silencing the RHBDF1 gene in MDA-MB-435 or 1483 xenograft tumors on athymic nude mice by using i.v. administered histidine-lysine polymer nanoparticle-encapsulated siRNA results in marked inhibition of tumor growth. Our findings indicate that RHBDF1 has a pivotal role in sustaining growth signals in epithelial cancer cells and thus may serve as a therapeutic target for treating epithelial cancers.

Targeting Cancer with Peptide RNAi Nanoplexes.

With the recent explosion of genomic information on the root causes of disease, there is an increased interest in nucleic acid therapeutics, including siRNA and gene therapy, all of which require delivery of highly charged nucleic acids from siRNA with a molecular weight of about 1.4 × 104 to plasmids with an approximate molecular weight of 2.0-3.0 × 106. This chapter describes the delivery of shRNA via plasmid or siRNA with a peptide-based carrier. We focus on the histidine-lysine peptide which serves as an example for other peptides and polymeric carrier systems. When the HK peptide and nucleic acids are mixed together and interact with one another through ionic and nonionic interactions, nanoplexes are formed. These nanoplexes, carrying either shRNA or siRNA that target oncogenes, provide promising options for the treatment of cancer. We describe methods of preparation and characterization of these nanoplexes using dynamic light scattering, zeta potential, and gel retardation assays. We also provide protocols for transfection in vitro and in vivo for these nanoplexes.

Advances in delivery systems for doxorubicin.

Doxorubicin is a widely used chemotherapy agent. Despite its utility, several adverse side effects, especially its irreversible cardiotoxicity and reversible nephrotoxicity, have prompted the development of liposomal carriers, many of which are FDA approved. Antitumor efficacies of approved liposome-Dox preparations can equal or exceed that of conventional doxorubicin. Because these liposomes carriers accumulate in solid tumor tissues via an enhanced permeation and retention (EPR) effect, these carriers have an improved safety profile. Nevertheless, a significant problem with the current drug delivery preparations of doxorubicin is a lack of efficacy toward tumors that exhibit multidrug resistance. In this review, we consider the development of drug delivery systems for doxorubicin, which improve the therapeutic window (efficacy and safety) and which address limitations of the current FDA-approved doxorubicin formulations.