Evaluation of Bacteriophage ϕ11 host recognition protein and its host-binding peptides for diagnosing/targeting of Saphylococcus aureus infections.

Evaluating the potential of using both synthetic and biological products as targeting agents for the diagnosis, imaging, and treatment of infections due to particularly antibiotic-resistant pathogens is important for controlling infections. We examined the interaction between Gp45, a receptor-binding protein of the ϕ11 lysogenic phage, and its host S. aureus, a common cause of nosocomial infections. Using molecular dynamics and docking simulations, we identified the peptides that bind to S. aureus wall teichoic acids via Gp45. We compared the binding affinity of Gp45 and the two highest-scoring peptide sequences (P1 and P3) and their scrambled forms using microscopy, spectroscopy, and ELISA. Our results revealed that rGp45 (recombinant Gp45) and chemically synthesized P1 had a higher binding affinity for S. aureus compared with all other peptides, with the exception of E. coli. Furthermore, rGp45 had a capture efficiency of over 86%; P1 had a capture efficiency of over 64%. Overall, our findings suggest that receptor-binding proteins such as rGp45, which provide a critical initiation of the phage life cycle for host adsorption, might play an important role in the diagnosis, imaging, and targeting of bacterial infections. Studying such proteins could accordingly enable the development of effective strategies for controlling infections.

An Asymmetric NIR-II Organic Fluorophore with an Ultra-Large Stokes Shift for Imaging-Guided and Targeted Phototherapy.

NIR-II imaging-guided phototherapy is an attractive, yet challenging, tumor treatment strategy. By monitoring the accumulation of phototherapy reagents at the tumor site through imaging and determining the appropriate therapy window, the therapeutic effect could be significantly improved. Probes with NIR-II (1000-1700 nm) fluorescence emission and a large Stokes shift hold great promise for fluorescence imaging with deep penetration, minimized self-quenching, and high spatiotemporal resolution. However, due to the lack of a suitable molecular framework, the design of a simple small-molecule dye with a large Stokes shift and NIR-II fluorescence emission has rarely been reported. Herein, we prepare an asymmetric D-π-A type NIR-II fluorescence probe (TBy). The probe is incapsulated in an amphiphilic polymer and modified with a fibronectin targeting peptide CREKA, which could recognize the fibrin-fibronectin complex overexpressed in multiple malignant tumors. The nanoparticles thus constructed (TByC-NPs) have maximum fluorescence emission at 1037 nm with a large Stokes shift of 426 nm, which is the largest Stokes shift among organic NIR-II fluorescent dyes reported in the literature. The TByC-NPs exhibit a good NIR-II imaging performance, active tumor targeting, and good photothermal and photodynamic capabilities. In vitro and in vivo studies verify that the TByC nanoplatform shows outstanding biocompatibility for NIR-II imaging-guided phototherapy and provides an excellent antitumor effect.

Small extracellular vesicle-mediated CRISPR-Cas9 RNP delivery for cardiac-specific genome editing.

Myocardial infarction (MI) is a major cause of morbidity and mortality worldwide. Although clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) gene editing holds immense potential for genetic manipulation, its clinical application is hindered by the absence of an efficient heart-targeted drug delivery system. Herein, we developed CRISPR-Cas9 ribonucleoprotein (RNP)-loaded extracellular vesicles (EVs) conjugated with cardiac-targeting peptide (T) for precise cardiac-specific genome editing. RNP complexes containing Cas9 and single guide RNA targeting miR-34a, an MI-associated molecular target, were loaded into EVs (EV@RNP). Gene editing by EV@RNP attenuated hydrogen peroxide-induced apoptosis in cardiomyocytes via miR-34a inhibition, evidenced by increased B-cell lymphoma 2 levels, decreased Bcl-2-associated X protein levels, and the cleavage of caspase-3. Additionally, to improve cardiac targeting in vivo, we used click chemistry to form functional T-EV@RNP by conjugating T peptides to EV@RNP. Consequently, T-EV@RNP-mediated miR-34a genome editing might exert a protective effect against MI, reducing apoptosis, ameliorating MI injury, and facilitating the recovery of cardiac function. In conclusion, the genome editing delivery system established by loading CRISPR/Cas9 RNP with cardiac-targeting EVs is a powerful approach for precise and tissue-specific gene therapy for cardiovascular disease.

Formulate Adaptive Biphasic Scaffold via Sequential Protein-Instructed Peptide Co-Assembly.

To ensure compositional consistency while mitigating potential immunogenicity for stem cell therapy, synthetic scaffolds have emerged as compelling alternatives to native extracellular matrix (ECM). Substantial progress has been made in emulating specific natural traits featuring consistent chemical compositions and physical structures. However, recapitulating the dynamic responsiveness of the native ECM involving chemical transitions and physical remodeling during differentiation, remains a challenging endeavor. Here, the creation of adaptive scaffolds is demonstrated through sequential protein-instructed molecular assembly, utilizing stage-specific proteins, and incorporating in situ assembly technique. The procedure is commenced by introducing a dual-targeting peptide at the onset of stem cell differentiation. In response to highly expressed integrins and heparan sulfate proteoglycans (HSPGs) on human mesenchymal stem cell (hMSC), the peptides assembled in situ, creating customized extracellular scaffolds that adhered to hMSCs promoting osteoblast differentiation. As the expression of alkaline phosphatase (ALP) and collagen (COL-1) increased in osteoblasts, an additional peptide is introduced that interacts with ALP, initiating peptide assembly and facilitating calcium phosphate (CaP) deposition. The growth and entanglement of peptide assemblies with collagen fibers efficiently incorporated CaP into the network resulting in an adaptive biphasic scaffold that enhanced healing of bone injuries.

Dendritic cell targeting peptide plus Salmonella FliCd flagellin fused outer membrane protein H (OmpH) demonstrated increased efficacy against infections caused by different Pasteurella multocida serogroups in mouse models.

As the major outer membrane protein (OMP) presents in the Pasteurella multocida envelope, OmpH was frequently expressed for laboratory assessments of its immunogenicity against P. multocida infections, but the results are not good. In this study, we modified OmpH with dendritic cell targeting peptide (Depeps) and/or Salmonella FliCd flagellin, and expressed three types of recombinant proteins with the MBP tag (rDepeps-FliC-OmpH-MBP, rDepeps-OmpH-MBP, rFliC-OmpH-MBP). Assessments in mouse models revealed that vaccination with rDepeps-FliC-OmpH-MBP, rDepeps-OmpH-MBP, or rFliC-OmpH-MBP induced significant higher level of antibodies as well as IFN-γ and IL-4 in murine sera than vaccination with rOmpH-MBP (P < 0.5). Vaccination with the three modified proteins also provided increased protection (rDepeps-FliC-OmpH-MBP, 70 %; rDepeps-OmpH-MBP, 50 %; rFliC-OmpH-MBP, 60 %) against P. multocida serotype D compared to vaccination with rOmpH-MBP (30 %). In mice vaccinated with different types of modified OmpHs, a significantly decreased bacterial strains were recovered from bloods, lungs, and spleens compared to rOmpH-MBP-vaccinated mice (P < 0.5). Notably, our assessments also demonstrated that vaccination with rDepeps-FliC-OmpH-MBP provided good protection against infections caused by a heterogeneous group of P. multocida serotypes (A, B, D). Our above findings indicate that modification with DCpep and Salmonella flagellin could be used as a promising strategy to improve vaccine effectiveness.

Characterisation of the cell and molecular biological effect of peptide-based daunorubicin conjugates developed for targeting pancreatic adenocarcinoma (PANC-1) cell line.

Pancreatic adenocarcinoma is one of the tumours with the worst prognosis, with a 5-year survival rate of 5-10%. Our aim was to find and optimise peptide-based drug conjugates with daunorubicin (Dau) as the cytotoxic antitumour agent. When conjugated with targeting peptides, the side effect profile and pharmacokinetics of Dau can be improved. The targeting peptide sequences (e.g. GSSEQLYL) we studied were originally selected by phage display. By Ala-scan technique, we identified that position 6 in the parental sequence (Dau=Aoa-LRRY-GSSEQLYL-NH2, ConjA) could be modified without the loss of antitumour activity (Dau=Aoa-LRRY-GSSEQAYL-NH2, Conj03: 14. 9% viability). Our results showed that the incorporation of p-chloro-phenylalanine (Dau=Aoa-LRRY-GSSEQF(pCl)YL-NH2, Conj16) further increased the antitumour potency (10-5 M: 9.7% viability) on pancreatic adenocarcinoma cells (PANC-1). We found that conjugates containing modified GSSEQLYL sequences could be internalised to PANC-1 cells and induce cellular senescence in the short term and subsequent apoptotic cell death. Furthermore, the cardiotoxic effect of Dau was markedly reduced in the form of peptide conjugates. In conclusion, Conj16 had the most effective antitumor activity on PANC-1 cells, which makes this conjugate promising for developing new targeted therapies without cardiotoxic effects.

Development of injectable upconversion nanoparticle-conjugated doxorubicin theranostics electrospun nanostructure for targeted photochemotherapy in breast cancer.

Nanotheranostic-based photochemotherapies with targeted drug delivery have considerably surfaced in cancer therapy. In the presented work, polyethyleneimine-coated upconversion nanoparticles were engineered to conjugate covalently with doxorubicin. Upconversion nanoparticles (UCNP)-Doxorubicin (DOX)/synthesized epidermal growth factor receptor-targeting peptide blended with polymer composite was electrospun and formulated as the injectable dosage form. The size of the UCNP and the nanofiber diameter were assessed as 26.75 ± 1.54 and 162 ± 2.82 nm, respectively. The optimized ratio of dopants resulted in UCNP photoluminescence with maximum emission intensity at around 800 nm upon 980 nm excitation wavelength. The paramagnetic nature of UCNPs and amide conjugation with the drug was confirmed analytically. The loading capacity of UCNP for doxorubicin was determined to be 54.56%, while nanofibers exhibited 98.74% capacity to encapsulate UCNP-DOX. The release profile of UCNP-DOX from nanofiber formulation ranged from sustained to controlled, with relative enhancement in acidic conditions. The nanofiber demonstrated good mechanical strength, robust swelling, and degradation rate. Biocompatibility tests showed more than 90% cell viability on L929 and NIH/3T3 cell lines with UCNP-DOX@NF/pep nanoformulation. The IC50 values of 2.15 ± 0.54, 2.87 ± 0.67, and 3.42 ± 0.45 µg/mL on MDA-MB-231, 4T1, and MCF-7 cancer cell line, respectively, with a significant cellular uptake, has been reported. The UCNP protruded a ≈62.7°C temperature rise within 5 min of 980 nm laser irradiation and a power density of 0.5 W cm-2. The nanoformulation induced reactive oxygen species of 65.67% ± 3.21% and apoptosis by arresting the cell cycle sub-G1 phase. The evaluation conveys the effectiveness of the developed injectable theranostic delivery system in cancer therapy.

Immunoprotection of FliBc chimeric fiber2 fusion proteins targeting dendritic cells against Fowl adenovirus serotype 4 infection.

Hepatitis-hydropericardium syndrome (HHS) is a highly fatal disease in chickens caused by the highly pathogenic fowl adenovirus serotype 4 (FAdV-4), which has severe economic consequences. The fiber2 protein exhibits excellent potential as a candidate for a subunit vaccination against FAdV-4. Despite having a high safety profile, subunit vaccines have low immunogenicity due to their lack of infectivity, which leads to low levels of immune response. As a vaccine adjuvant, Salmonella flagellin possesses the potential to augment the immunological response to vaccinations. Additionally, a crucial strategy for enhancing vaccine efficacy is efficient presentation of immune antigens to dendritic cells (DC) for targeted vaccination. In this study, we designed FAdV-4-fiber2 protein, and a recombinant protein called FliBc-fiber2-SP which based on FAdV-4-fiber2 protein, was generated using the gene sequence FliBc, which retains only the conserved sequence at the amino and carboxyl termini of the flagellin B subunit, and a short peptide SPHLHTSSPWER (SP), which targets chicken bone marrow-derived DC. They were separately administered via intramuscular injection to 14-day-old specific pathogen-free (SPF) chickens, and their immunogenicity was compared. At 21 d postvaccination (dpv), it was found that the FliBc-fiber2-SP recombinant protein elicited significantly higher levels of IgG antibodies and conferred a vaccine protection rate of up to 100% compared to its counterpart fiber2 protein. These results suggest that the DC-targeted peptide fusion strategy for flagellin chimeric antigen construction can effectively enhance the immune protective efficacy of antigen proteins.

In vivo therapy of osteosarcoma using anion transporters-based supramolecular drugs.

BACKGROUND: Osteosarcoma represents a serious clinical challenge due to its widespread genomic alterations, tendency for drug resistance and distant metastasis. New treatment methods are urgently needed to address those treatment difficulties in osteosarcoma to improve patient prognoses. In recent years, small-molecule based anion transporter have emerged as innovative and promising therapeutic compound with various biomedical applications. However, due to a lack of efficient delivery methods, using ion transporters as therapeutic drugs in vivo remains a major challenge. RESULT: Herein, we developed self-assembled supramolecular drugs based on small-molecule anion transporters, which exhibited potent therapeutic effect towards osteosarcoma both in vitro and in vivo. The anion transporters can disrupt intracellular ion homeostasis, inhibit proliferation, migration, epithelial-mesenchymal transition process, and lead to osteosarcoma cell death. RNA sequencing, western blot and flow cytometry indicated reprogramming of HOS cells and induced cell death through multiple pathways. These pathways included activation of endoplasmic reticulum stress, autophagy, apoptosis and cell cycle arrest, which avoided the development of drug resistance in osteosarcoma cells. Functionalized with osteosarcoma targeting peptide, the assembled supramolecular drug showed excellent targeted anticancer therapy against subcutaneous xenograft tumor and lung metastasis models. Besides good tumor targeting capability and anti-drug resistance, the efficacy of the assembly was also attributed to its ability to regulate the tumor immune microenvironment in vivo. CONCLUSIONS: In summary, we have demonstrated for the first time that small-molecule anion transporters are capable of killing osteosarcoma cells through multiple pathways. The assemblies, OTP-BP-L, show excellent targeting and therapeutic effect towards osteosarcoma tumors. Furthermore, the supramolecular drug shows a strong ability to regulate the tumor immune microenvironment in vivo. This work not only demonstrated the biomedical value of small-molecule anion transporters in vivo, but also provided an innovative approach for the treatment of osteosarcoma.

Microarray-Based CD38 Peptide Probe Screening for Multiple Myeloma Imaging.

Assessing CD38 expression in vivo has become a significant element in multiple myeloma (MM) therapy, as it can be used to detect lesions and forecast the effectiveness of treatment. Accurate diagnosis requires a multifunctional, high-throughput probe screening platform to develop molecular probes for tumor-targeted multimodal imaging and treatment. Here, we investigated a microarray chip-based strategy for high-throughput screening of peptide probes for CD38. We obtained two new target peptides, CA-1 and CA-2, from a 105 peptide library with a dissociation constant (KD) of 10-7 M. The specificity and affinity of the target peptides were confirmed at the molecular and cellular levels. Peptide probes were labeled with indocyanine green (ICG) dye and 68Ga-DOTA, which were injected into a CD38-positive Ramos tumor-bearing mouse via its tail vein, and small animal fluorescence and positron emission tomography (PET) imaging showed that the peptide probes could show specific enrichment in the tumor tissue. Our study shows that a microchip-based screening of peptide probes can be used as a promising imaging tool for MM diagnosis.

Protein and peptide-based renal targeted drug delivery systems.

Renal diseases have become an increasingly concerned public health problem in the world. Kidney-targeted drug delivery has profound transformative potential on increasing renal efficacy and reducing extra-renal toxicity. Protein and peptide-based kidney targeted drug delivery systems have garnered more and more attention due to its controllable synthesis, high biocompatibility and low immunogenicity. At the same time, the targeting methods based on protein/peptide are also abundant, including passive renal targeting based on macromolecular protein and active targeting mediated by renal targeting peptide. Here, we review the application and the drug loading strategy of different proteins or peptides in targeted drug delivery, including the ferritin family, albumin, low molecular weight protein (LMWP), different peptide sequence and antibodies. In addition, we summarized the factors influencing passive and active targeting in drug delivery system, the main receptors related to active targeting in different kidney diseases, and a variety of nano forms of proteins based on the controllable synthesis of proteins.

Cardiac-Targeting Peptide: From Discovery to Applications.

Despite significant strides in prevention, diagnosis, and treatment, cardiovascular diseases remain the number one cause of mortality in the United States, with rates climbing at an alarming rate in the developing world. Targeted delivery of therapeutics to the heart has been a lofty goal to achieve with strategies ranging from direct intra-cardiac or intra-pericardial delivery, intra-coronary infusion, to adenoviral, lentiviral, and adeno-associated viral vectors which have preference, if not complete cardio-selectivity, for cardiac tissue. Cell-penetrating peptides (CPP) are 5-30-amino-acid-long peptides that are able to breach cell membrane barriers while carrying cargoes up to several times their size, in an intact functional form. Identified nearly three decades ago, the first of these CPPs came from the HIV coat protein transactivator of transcription. Although a highly efficient CPP, its clinical utility is limited by its robust ability to cross any cell membrane barrier, including crossing the blood-brain barrier and transducing neuronal tissue non-specifically. Several strategies have been utilized to identify cell- or tissue-specific CPPs, one of which is phage display. Using this latter technique, we identified a cardiomyocyte-targeting peptide (CTP) more than a decade ago, a finding that has been corroborated by several independent labs across the world that have utilized CTP for a myriad of different purposes in pre-clinical animal models. The goal of this publication is to provide a comprehensive review of the identification, validation, and application of CTP, and outline its potential in diagnostic and therapeutic applications especially in the field of targeted RNA interference.

Targeted drug delivery into glial scar using CAQK peptide in a mouse model of multiple sclerosis.

In multiple sclerosis, lesions are formed in various areas of the CNS, which are characterized by reactive gliosis, immune cell infiltration, extracellular matrix changes and demyelination. CAQK peptide (peptide sequence: cysteine-alanine-glutamine-lysine) was previously introduced as a targeting peptide for the injured site of the brain. In the present study, we aimed to develop a multifunctional system using nanoparticles coated by CAQK peptide, to target the demyelinated lesions in animal model of multiple sclerosis. We investigated the binding of fluorescein amidite-labelled CAQK and fluorescein amidite-labelled CGGK (as control) on mouse brain sections. Then, the porous silicon nanoparticles were synthesized and coupled with fluorescein amidite-labelled CAQK. Five days after lysolecithin-induced demyelination, male mice were intravenously injected with methylprednisolone-loaded porous silicon nanoparticles conjugated to CAQK or the same amount of free methylprednisolone. Our results showed that fluorescein amidite-labelled CAQK recognizes demyelinated lesions in brain sections of animal brains injected with lysolecithin. In addition, intravenous application of methylprednisolone-loaded nanoparticle porous silicon conjugated to CAQK at a single dose of 0.24 mg reduced the levels of microglial activation and astrocyte reactivation in the lesions of mouse corpus callosum after 24 and 48 h. No significant effect was observed following the injection of the same dose of free methylprednisolone. CAQK seems a potential targeting peptide for delivering drugs or other biologically active chemicals/reagents to the CNS of patients with multiple sclerosis. Low-dose methylprednisolone in this targeted drug delivery system showed significant beneficial effect.

PET imaging with [68Ga]-labeled TGFß-targeting peptide in a mouse PANC-1 tumor model.

Purpose: Transforming growth factor ß (TGFß) is upregulated in many types of tumors and plays important roles in tumor microenvironment construction, immune escape, invasion, and metastasis. The therapeutic effect of antibodies and nuclide-conjugated drugs targeting TGFß has not been ideal. Targeting TGFß with small-molecule or peptide carriers labeled with diagnostic/therapeutic nuclides is a new development direction. This study aimed to explore and confirm the imaging diagnostic efficiency of TGFß-targeting peptide P144 coupled with [68Ga] in a PANC-1 tumor model. Procedures: TGFß-targeting inhibitory peptide P144 with stable activity was prepared through peptide synthesis and screening, and P144 was coupled with biological chelator DOTA and labeled with radionuclide [68Ga] to achieve a stable TGFß-targeting tracer [68Ga]Ga-P144. This tracer was first used for positron emission tomography (PET) molecular imaging study of pancreatic cancer in a mouse PANC-1 tumor model. Results: [68Ga]Ga-P144 had a high targeted uptake and relatively long uptake retention time in tumors and lower uptakes in non-target organs and backgrounds. Target pre-blocking experiment with the cold drug P144-DOTA demonstrated that the radioactive uptake with [68Ga]Ga-P144 PET in vivo, especially in tumor tissue, had a high TGFß-targeting specificity. [68Ga]Ga-P144 PET had ideal imaging efficiency in PANC-1 tumor-bearing mice, with high specificity in vivo and good tumor-targeting effect. Conclusion: [68Ga]Ga-P144 has relatively high specificity and tumor-targeted uptake and may be developed as a promising diagnostic tool for TGFß-positive malignancies.

Engineered small extracellular vesicle-mediated NOX4 siRNA delivery for targeted therapy of cardiac hypertrophy.

Small-interfering RNA (siRNA) therapy is considered a powerful therapeutic strategy for treating cardiac hypertrophy, an important risk factor for subsequent cardiac morbidity and mortality. However, the lack of safe and efficient in vivo delivery of siRNAs is a major challenge for broadening its clinical applications. Small extracellular vesicles (sEVs) are a promising delivery system for siRNAs but have limited cell/tissue-specific targeting ability. In this study, a new generation of heart-targeting sEVs (CEVs) has been developed by conjugating cardiac-targeting peptide (CTP) to human peripheral blood-derived sEVs (PB-EVs), using a simple, rapid and scalable method based on bio-orthogonal copper-free click chemistry. The experimental results show that CEVs have typical sEVs properties and excellent heart-targeting ability. Furthermore, to treat cardiac hypertrophy, CEVs are loaded with NADPH Oxidase 4 (NOX4) siRNA (siNOX4). Consequently, CEVs@siNOX4 treatment enhances the in vitro anti-hypertrophic effects by CEVs with siRNA protection and heart-targeting ability. In addition, the intravenous injection of CEVs@siNOX4 into angiotensin II (Ang II)-treated mice significantly improves cardiac function and reduces fibrosis and cardiomyocyte cross-sectional area, with limited side effects. In conclusion, the utilization of CEVs represents an efficient strategy for heart-targeted delivery of therapeutic siRNAs and holds great promise for the treatment of cardiac hypertrophy.

Cardiomyocyte-Targeting Peptide to Deliver Amiodarone.

BACKGROUND: Amiodarone is underutilized due to significant off-target toxicities. We hypothesized that targeted delivery to the heart would lead to the lowering of the dose by utilizing a cardiomyocyte-targeting peptide (CTP), a cell-penetrating peptide identified by our prior phage display work. METHODS: CTP was synthesized thiolated at the N-terminus, conjugated to amiodarone via Schiff base chemistry, HPLC purified, and confirmed with MALDI/TOF. The stability of the conjugate was assessed using serial HPLCs. Guinea pigs (GP) were injected intraperitoneally daily with vehicle (7 days), amiodarone (7 days; 80 mg/kg), CTP-amiodarone (5 days; 26.3 mg/kg), or CTP (5 days; 17.8 mg/kg), after which the GPs were euthanized, and the hearts were excised and perfused on a Langendorff apparatus with Tyrode's solution and blebbistatin (5 µM) to minimize the contractions. Voltage (RH237) and Ca2+-indicator dye (Rhod-2/AM) were injected, and fluorescence from the epicardium split and was captured by two cameras at 570-595 nm for the cytosolic Ca2+ and 610-750 nm wavelengths for the voltage. Subsequently, the hearts were paced at 250 ms with programmed stimulation to measure the changes in the conduction velocities (CV), action potential duration (APD), and Ca2+ transient durations at 90% recovery (CaTD90). mRNA was extracted from all hearts, and RNA sequencing was performed with results compared to the control hearts. RESULTS: The CTP-amiodarone remained stable for up to 21 days at 37 °C. At ~1/15th of the dose of amiodarone, the CTP-amiodarone decreased the CV in hearts significantly compared to the control GPs (0.92 ± 0.05 vs. 1.00 ± 0.03 ms, p = 0.0007), equivalent to amiodarone alone (0.87 ± 0.08 ms, p = 0.0003). Amiodarone increased the APD (192 ± 5 ms vs. 175 ± 8 ms for vehicle, p = 0.0025), while CTP-amiodarone decreased it significantly (157 ± 16 ms, p = 0.0136), similar to CTP alone (155 ± 13 ms, p = 0.0039). Both amiodarone and CTP-amiodarone significantly decreased the calcium transients compared to the controls. CTP-amiodarone and CTP decreased the CaTD90 to an extent greater than amiodarone alone (p < 0.001). RNA-seq showed that CTP alone increased the expression of DHPR and SERCA2a, while it decreased the expression of the proinflammatory genes, NF-kappa B, TNF-α, IL-1ß, and IL-6. CONCLUSIONS: Our data suggest that CTP can deliver amiodarone to cardiomyocytes at ~1/15th the total molar dose of the amiodarone needed to produce a comparable slowing of CVs. The ability of CTP to decrease the AP durations and CaTD90 may be related to its increase in the expression of Ca-handling genes, which merits further study.

Metal-Organic Frameworks Nucleated by Silk Fibroin and Modified with Tumor-Targeting Peptides for Targeted Multimodal Cancer Therapy.

Multimodal therapy requires effective drug carriers that can deliver multiple drugs to specific locations in a controlled manner. Here, the study presents a novel nanoplatform constructed using zeolitic imidazolate framework-8 (ZIF-8), a nanoscale metal-organic framework nucleated under the mediation of silk fibroin (SF). The nanoplatform is modified with the newly discovered MCF-7 breast tumor-targeting peptide, AREYGTRFSLIGGYR (AR peptide). Indocyanine green (ICG) and doxorubicin (DOX) are loaded onto the nanoplatform with high drug encapsulation efficiency (>95%). ICG enables the resultant nanoparticles (NPs), called AR-ZS/ID-P, to release reactive oxygen species for photodynamic therapy (PDT) and heat for photothermal therapy (PTT) under near-infrared (NIR) irradiation, promoting NIR fluorescence and thermal imaging to guide DOX-induced chemotherapy. Additionally, the controlled release of both ICG and DOX at acidic tumor conditions due to the dissolution of ZIF-8 provides a drug-targeting mechanism in addition to the AR peptide. When intravenously injected, AR-ZS/ID-P NPs specifically target breast tumors and exhibit higher anticancer efficacy than other groups through ICG-enabled PDT and PTT and DOX-derived chemotherapy, without inducing side effects. The results demonstrate that AR-ZS/ID-P NPs are a promising multimodal theranostic nanoplatform with maximal therapeutic efficacy and minimal side effects for targeted and controllable drug delivery.

Conjugation of amiodarone to a novel cardiomyocyte cell penetrating peptide for potential targeted delivery to the heart.

Modern medicine has developed a myriad of therapeutic drugs against a wide range of human diseases leading to increased life expectancy and better quality of life for millions of people. Despite the undeniable benefit of medical advancements in pharmaceutical technology, many of the most effective drugs currently in use have serious limitations such as off target side effects resulting in systemic toxicity. New generations of specialized drug constructs will enhance targeted therapeutic efficacy of existing and new drugs leading to safer and more effective treatment options for a variety of human ailments. As one of the most efficient drugs known for the treatment of cardiac arrhythmia, Amiodarone presents the same conundrum of serious systemic side effects associated with long term treatment. In this article we present the synthesis of a next-generation prodrug construct of amiodarone for the purpose of advanced targeting of cardiac arrhythmias by delivering the drug to cardiomyocytes using a novel cardiac targeting peptide, a cardiomyocyte-specific cell penetrating peptide. Our in vivo studies in guinea pigs indicate that cardiac targeting peptide-amiodarone conjugate is able to have similar effects on calcium handling as amiodarone at 1/15th the total molar dose of amiodarone. Further studies are warranted in animal models of atrial fibrillation to show efficacy of this conjugate.

CXCR4 Targeting Nanoplatform for Transcriptional Activation of Latent HIV-1 Infected T Cells.

Antiretroviral drugs are limited in their ability to target latent retroviral reservoirs in CD4+ T cells, highlighting the need for a T cell-targeted drug delivery system that activates the transcription of inactivated viral DNA in infected cells. Histone deacetylase inhibitors (HDACi) disrupt chromatin-mediated silencing of the viral genome and are explored in HIV latency reversal. But single drug formulations of HDACi are insufficient to elicit therapeutic efficacy, warranting combination therapy. Furthermore, protein kinase C activators (PKC) have shown latency reversal activity in HIV by activating the NF-κB signaling pathway. Combining HDACi (SAHA) with PKC (PMA) activators enhances HIV reservoir activation by promoting chromatin decondensation and subsequent transcriptional activation. In this study, we developed a mixed nanomicelle (PD-CR4) drug delivery system for simultaneous targeting of HIV-infected CD4+ T cells with two drugs, suberoylanilide hydroxamic acid (SAHA) and phorbol 12-myristate 13-acetate (PMA). SAHA is a HDACi that promotes chromatin decondensation, while PMA is a PKC agonist that enhances transcriptional activation. The physicochemical properties of the formulated PD-CR4 nanoparticles were characterized by NMR, CMC, DLS, and TEM analyses. Further, we investigated in vitro safety profiles, targeting efficacy, and transcriptional activation of inactivated HIV reservoir cells. Our results suggest that we successfully prepared a targeted PD system with dual drug loading. We have compared latency reversal efficacy of a single drug nanoformulation and combination drug nanoformulation. Final PD-SP-CR4 successfully activated infected CD4+ T cell reservoirs and showed enhanced antigen release from HIV reservoir T cells, compared with the single drug treatment group as expected. To summarize, our data shows PD-SP-CR4 has potential T cell targeting efficiency and efficiently activated dormant CD4+ T cells. Our data indicate that a dual drug-loaded particle has better therapeutic efficacy than a single loaded particle as expected. Hence, PD-CR4 can be further explored for HIV therapeutic drug delivery studies.

A dual-targeting peptide for glioblastoma screened by phage display peptide library biopanning combined with affinity-adaptability analysis.

The obstruction of blood-brain barrier (BBB) and the poor specific targeting are still the major obstacles and challenges of targeted nano-pharmaceutical therapy for glioblastoma (GBM) up to now. It is critical to find appropriate targeting ligands that can effectively mediate the nano-pharmaceuticals to penetrate brain capillary endothelial cells (BCECs) and then specifically bind to glioblastoma cells (GCs). Herein, a dual-targeting ligand for GBM was screened by the combination of phage display peptide library biopanning and affinity-adaptability analysis. Based on the acquisition of sub-library of peptide which exhibited the specific affinity to both BCECs and GCs, a comparison parameter of relative affinity was deliberately introduced to evaluate the relative affinity of candidate peptides to U251-MG cells and bEnd.3 cells. The optimized WTW peptide (sequenced as WTWEYTK) was provided with a high relative affinity (RU/B = 2.44), implying that its high affinity to U251-MG cells and moderate affinity to bEnd.3 cells might synergistically promote its receptor-mediated internalization and transport, the dissociation from bEnd.3, and the binding to U251-MG. The results of BBB model trials in vitro showed that the BBB penetration efficiency and GBM accumulation of WTW peptide were significantly higher than those of WSL peptide, GNH peptide, and REF peptide. Results of orthotopic GBM xenograft model assays in vivo also indicated that WTW peptide had successfully penetrated the BBB and improved accumulation in GBM. The screened WTW peptide might be the potential dual-targeting ligand to motivate the advancement of GBM targeted therapy.