Microsphere-Structured Protein Hydrogel Dielectrics for Capacitive Wearable Sensors.

The desire for healthy living has created a crucial need for portable flexible health-monitoring devices based on biomaterials. Toward this end, we report a microsphere-structured hydrogel that uses bovine serum albumin (BSA) as a dielectric layer for capacitive pressure sensors. We developed a theoretical model that describes how stacking dielectric layers of spheres affects the performance of capacitive sensors. We also prepared a prototype sensor featuring the unique microsphere structure to create capacitive sensors with high sensitivity (360.91 strain sensitivity), excellent cyclical stability, and a long service life (over 5000 stretching-compression cycles). Furthermore, the design of the hydrogel sensor allows for easy integration into fabrics to create devices such as smart wristbands, which can collect a diverse range of health data. Thus, BSA-hydrogel-based sensors not only provide safe wearable devices but also advance the performance of high-sensitivity capacitive sensors.

Regulation of cerebral blood flow boosts precise brain targeting of vinpocetine-derived ionizable-lipidoid nanoparticles.

Despite advances in active drug targeting for blood-brain barrier penetration, two key challenges persist: first, attachment of a targeting ligand to the drug or drug carrier does not enhance its brain biodistribution; and second, many brain diseases are intricately linked to microcirculation disorders that significantly impede drug accumulation within brain lesions even after they cross the barrier. Inspired by the neuroprotective properties of vinpocetine, which regulates cerebral blood flow, we propose a molecular library design centered on this class of cyclic tertiary amine compounds and develop a self-enhanced brain-targeted nucleic acid delivery system. Our findings reveal that: (i) vinpocetine-derived ionizable-lipidoid nanoparticles efficiently breach the blood-brain barrier; (ii) they have high gene-loading capacity, facilitating endosomal escape and intracellular transport; (iii) their administration is safe with minimal immunogenicity even with prolonged use; and (iv) they have potent pharmacologic brain-protective activity and may synergize with treatments for brain disorders as demonstrated in male APP/PS1 mice.

Design and function of lignin/silk fibroin-based multilayer water purification membranes for dye adsorption.

The treatment of dye wastewater poses a significant challenge to the sewage recycling industries. However, the reduction of secondary pollution resulting from the membrane residues, to maintain high performance, remains a considerable obstacle. A novel approach for the fabrication of multilayer nanofiber structures using a layer-by-layer electrostatic spinning technique with biological materials was reported in this study. Incorporating the chemical adsorption advantages of lignin nanofiber and the physical adsorption advantages of silk fibroin (SF) nanofiber enabled the full realization of excellent dye interception performance. A comparative analysis was conducted on the lignin derived from eucalyptus, pine, and straw to determine the most suitable option. Notably, eucalyptus lignin exhibited superior antimicrobial properties. The adsorption of crystal violet by eucalyptus lignin/SF membrane was consistent with the Freundlich isotherm model and the pseudo-second-order kinetic model, revealing a chemisorption mechanism involving Π-Π conjugation, hydrogen bonding, and the binding of anions and cations. The lignin/SF membrane exhibited a retention rate exceeding 99.5 % for crystal violet, methylene blue, and brilliant green dyes. Furthermore, it demonstrated efficacy in retaining heavy metal ions, including cadmium and copper. The original biomass material imparts the property of rapid degradation to a multilayer membrane that can be used as an effective and eco-friendly water purification material.

Breaking Spatiotemporal Barriers of Immunogenic Chemotherapy via an Endoplasmic Reticulum Membrane-Assisted Liposomal Drug Delivery.

Immunogenic chemotherapy is a promising approach in cancer treatment, but the number of drugs capable of inducing immunogenic cell death is limited, and chronic immunogenic exposure can delay antitumor immune response and be counteracted by immunosuppressive factors. In this study, we used single-cell and multilevel analyses to highlight the critical importance of the first exposure to calreticulin (CRT) in eliciting immunogenicity. We then developed the ERASION (endoplasmic reticulum (ER) membrane to assist (AS) the presentation of intrinsic onco-immunogenicity (ION)) strategy, leveraging the high expression of functional proteins, including CRT, on the ER membrane. ER membrane-coated liposome (ER@PLip) was able to target the tumor and immune effectors and promoted dendritic cell maturation and T cell infiltration. This enabled eliciting an immunogenic effect from a nonimmunogenic chemotherapeutic drug. By utilizing the ER membrane-associated STING protein, ERASION enabled activating the STING pathway and the generation of adaptive antitumor immunity. This study presents a potential universal platform for integrating traditional chemotherapy and therapeutic modalities.

Physical- and Chemical-Dually ROS-Responsive Nano-in-Gel Platforms with Sequential Release of OX40 Agonist and PD-1 Inhibitor for Augmented Combination Immunotherapy.

Combination immunotherapy synergizing the PD-1 blockade with OX40 agonism has become a research hotspot, due to its enormous potential to overcome the restricted clinical objective response suffered by monotherapy. Questions of timing and sequence have been important aspects of immunotherapies when considering immunologic mechanisms; however, most of the time the straightforward additive approach was taken. Herein, our work is the first to investigate an alternative timing of aOX40 and aPD-1 treatment in melanoma-bearing mice, and it demonstrates that sequential administration (aOX40 first, then aPD-1 following) provided superior antitumor benefits than concurrent treatment. Based on that, to further avoid the limits suffered by solution forms, we adopted pharmaceutical technologies to construct an in situ-formed physical- and chemical-dually ROS-responsive nano-in-gel platform to implement sequential and prolonged release of aPD-1 and aOX40. Equipped with these advantages, the as-prepared (aPD-1NCs&aOX40)@Gels elicited augmented combination immunity and achieved great eradication of both primary and distant melanoma tumors in vivo.

Overcoming the On-Target Toxicity in Antibody-Mediated Therapies via an Indirect Active Targeting Strategy.

Antibody-based therapies could be led astray when target receptors are expressed on nontarget sites, and the on-target toxicity poses critical challenges to clinical applications. Here, a biomimetic indirect active targeting (INTACT) strategy is proposed based on receptor expression disparities between nontarget sites and the targets. By prebinding the antibodies using cell membrane vesicles with appropriate receptor expressions, the INTACT strategy could filter out the interactions on nontarget sites due to their inferior receptor expression, whereas ensure on-demand release at the targets by competitive binding. The strategy is verified on CD47 antibody, realizing drastic alleviation of its clinically concerned hematotoxicity on a series of animal models including humanized patient-derived xenograft platforms, accompanied by preferable therapeutic effects. Furthermore, the INTACT strategy proves extensive applicability for various systems including antibody, antibody-drug conjugate, and targeted delivery systems, providing a potential platform refining the specificity for frontier antibody-related therapies.

Smart drug delivery systems for precise cancer therapy.

Nano-drug delivery strategies have been highlighted in cancer treatment, and much effort has been made in the optimization of bioavailability, biocompatibility, pharmacokinetics profiles, and in vivo distributions of anticancer nano-drug delivery systems. However, problems still exist in the delicate balance between improved anticancer efficacy and reduced toxicity to normal tissues, and opportunities arise along with the development of smart stimuli-responsive delivery strategies. By on-demand responsiveness towards exogenous or endogenous stimulus, these smart delivery systems hold promise for advanced tumor-specificity as well as controllable release behavior in a spatial-temporal manner. Meanwhile, the blossom of nanotechnology, material sciences, and biomedical sciences has shed light on the diverse modern drug delivery systems with smart characteristics, versatile functions, and modification possibilities. This review summarizes the current progress in various strategies for smart drug delivery systems against malignancies and introduces the representative endogenous and exogenous stimuli-responsive smart delivery systems. It may provide references for researchers in the fields of drug delivery, biomaterials, and nanotechnology.

Precise Delivery of Nanomedicines to M2 Macrophages by Combining "Eat Me/Don't Eat Me" Signals and Its Anticancer Application.

Targeted delivery of nanomedicines to M2 tumor-associated macrophages (TAMs) has been proposed to reduce tumor promotion and enhance the efficacy of anticancer therapy. However, upregulated receptors on M2 TAMs are also expressed on M1 TAMs and other macrophages in normal tissues. Therefore, improving targeting specificity remains a key challenge. Here, we developed a precise M2 TAM-targeted delivery system using "eat-me" and "don't-eat-me" signals. A CD47-derived self-peptide ligand (don't-eat-me signal) and galactose ligand (eat-me signal) were introduced on liposomes. Cleavable phospholipid-polyethylene glycol was covered on the surface and could combine with the self-peptide to inhibit macrophage recognition even after immunoglobulin M adsorption and protect galactose from hepatic clearance to prolong the circulation time and promote the accumulation of liposomes in tumors. This detachable polymer can be removed by the redox microenvironment upon transcytosis through the tumor endothelium and re-expose the self-peptide and galactose. The self-peptide highly reduced M1 macrophage phagocytosis, and the galactose ligand enhanced the interaction between the liposomes and M2 macrophages. Thus, the modified liposomes enabled specific recognition of M1/M2 TAMs. In vitro evidence revealed reduced endocytosis of the liposomes by M1 macrophages. Moreover, in vivo studies demonstrated that doxorubicin-loaded liposomes efficiently eliminated M2 TAMs but did not affect M1 TAMs, enhancing the potency of the antitumor therapy. Collectively, our results demonstrate the potential of combining active escape and active targeting for precisely delivering a drug of interest to M2 macrophages and suggest its application in anticancer therapy.

A Programmed Cell-Mimicking Nanoparticle Driven by Potato Alkaloid for Targeted Cancer Chemoimmunotherapy.

Membrane camouflaged-nanoparticles (CM-NPs) have been exploited to inherit desired functionalities from source cells. Despite those advantages, membrane cloak may play a "double-edged sword" role in tumor-targeting therapy, as the intact membrane coating may hinder function-exertion of loaded drugs after reaching predetermined site. Therefore, further optimization of CM-NPs is still needed to enhance their delivery efficiency. Herein, natural product, Solamargine (SM), a cholesterol-affiliative amphiphilic potato alkaloid is first applied as core component of "inner core," to design a cell-mimicking "core-shell" nanoparticle (RBC-SLip) with acid-responsive off-coating properties for tumor-targeted therapy. Owing to red blood cell membrane (RBCm)-derived outer coating, it circulates stably in physiological conditions. While it would undergo an off-coating morphological change in response to acid stimuli in tumor microenvironment (TME), afterwards, the resulting off-coating liposome (SLip) shows active tumor-targeting and endosomal escape abilities, thus contributing to superior antitumor efficacy. In addition, SM also possesses natural TME-modulating ability; therefore, RBC-SLip can synergize with the PD1/PD-L1 blockade immunotherapy when encapsulated with PTX to achieve enhanced chemoimmunotherapy. The off-coating strategy developed by natural products SM, provide a brand-new perspective to optimize CM-NPs, and it also embodies application value of "unification of medicines and excipients" of natural products.

Engineered liposomes targeting the gut-CNS Axis for comprehensive therapy of spinal cord injury.

Effective curative therapies for spinal cord injury (SCI), which is often accompanied by intestinal complications, are lacking. Potential therapeutic targets include astrocytes and their enteric nervous system counterpart, enteric glial cells (EGCs). Based on shared biomarkers and similar functions of both cell types, we designed an orally administered targeted delivery system in which the neuropeptide apamin, stabilized by sulfur replacement with selenium, was adopted as a targeting moiety, and the liposome surface was protected with a non-covalent cross-linked chitosan oligosaccharide lactate layer. The system effectively permeated through oral absorption barriers, targeted local EGCs and astrocytes after systemic circulation, allowing for comprehensive SCI therapy. Given the involvement of the gut-organ axis in a growing number of diseases, our research may shed light on new aspects of the oral administration route as a bypass for multiple interventions and targeted therapy.

Strategies of Drug Delivery for Deep Fungal Infection: A Review.

The deep fungal infection poses serious threats to human health, mainly due to the increase in the number of immunocompromised individuals. Current first-line antifungal agents such as Amphotericin B, Fluconazole and Itraconazole, may decrease the severity of fungal infection to some extent, but the poor drug bioavailability, drug toxicity and poor water solubility seriously restrict their clinical utility. This review focuses on the study of drug delivery strategies for the treatment of deep fungal infections. We summarize the drug delivery strategies recently reported for the treatment of deep fungal infection, and explain each part with research examples. We discuss the use of pharmaceutical approaches to improve the physicochemical properties of the antifungal drugs to provide a basis for the clinical application of antifungal drugs. We then highlight the strategies for targeting drug delivery to the infection sites of fungi and fungal surface moieties, which have the potential to get developed as clinically relevant targeted therapies against deep fungal infections. It is worth noting that the current research on fungal infections still lags behind the research on other pathogens, and the drug delivery strategy for the treatment of deep fungal infections is far from meeting the treatment needs. Therefore, we envision the potential strategies inspired by the treatment of diseases with referential pathology or pathophysiology, further enriching the delivery of antifungal agents, providing references for basic research of fungal infections. Lay Summary: The deep fungal infections pose serious threats to the health of immunodeficiency patients. It is worth noting that the current research on fungi is still lagging behind that on other pathogens. The drug delivery strategies for the treatment of deep fungal infections are far from meeting the treatment needs. We summarize the recently reported drug delivery strategies for treating deep fungal infection, and envision the potential strategies to further enrich the delivery of antifungal agents.

Anisotropic active ligandations in siRNA-Loaded hybrid nanodiscs lead to distinct carcinostatic outcomes by regulating nano-bio interactions.

Active targeting modification is one of the foremost nanomedicine strategies for the efficacy improvement. Compared to the homogeneous ligandation on spherical nanocarriers, non-spherical nanomedicines usually make the ligand modification more complicated. The modified ligands always exhibit anisotropy and heterogeneity. However, there is very little systematic study on these diversified anisotropic modifications. The efficacy difference and underlying mechanism were still unclear. Here, we separately fabricated hybrid nanodiscs (NDs) conjugated with cRGD on the edge and plane surfaces to engineer two anisotropic targeting nanocarriers (E-cRGD-NDs and P-cRGD-NDs, respectively) for gene delivery. The ligand anisotropy endowed NDs with diversified cellular interactions, and caused different efficacies between E-cRGD-NDs and P-cRGD-NDs. Of note, E-cRGD-NDs showed significant superiority in siRNA loading, cellular uptake, silence efficiency, protein expression and even in vivo efficacy. The mechanism investigation revealed the functional anisotropy specifically for E-cRGD-NDs. The edge modification of cRGD efficiently separated the targeting and siRNA loading domains, maximizing their respective functions. These findings reflected the unique effect of ligand anisotropy, also provided a new strategy for the targeting screening of extensive nanomedicines.

Establishment and Application of a Dual TaqMan Real-Time PCR Method for Proteus Mirabilis and Proteus Vulgaris.

Proteus species are common opportunistic bacteria and foodborne pathogens. The proper detection of Proteus can effectively reduce the occurrence of food-borne public health events. Proteus mirabilis and Proteus vulgaris are the two most important pathogens in the Proteus genus. In this study, a dual TaqMan Real-Time PCR method was established to simultaneously detect and distinguish P. mirabilis and P. vulgaris in samples. The method exhibited good specificity, stability, and sensitivity. Specifically, the minimum detection concentrations of P. mirabilis and P. vulgaris in pure bacterial cultures were 6.08 × 102 colony forming units (CFU)/ml and 4.46 × 102 CFU/ml, respectively. Additionally, the minimum detectable number of P. mirabilis and P. vulgaris in meat and milk was 103 CFU/g. In addition, the method can be used to distinguish between strains of P. mirabilis and P. vulgaris within two hours. Overall, it is a sensitive, easy-to-use, and practical test for the identification and classification of Proteus in food.

Overcoming the Reticuloendothelial System Barrier to Drug Delivery with a "Don't-Eat-Us" Strategy.

Overcoming the reticuloendothelial system (RES) has long been a vital challenge to nanoparticles as drug carriers. Modification of nanoparticles with polyethylene glycol helps them avoid clearance by macrophages but also suppresses their internalization by target cells. To overcome this paradox, we developed an RES-specific blocking system utilizing a "don't-eat-us" strategy. First, a CD47-derived, enzyme-resistant peptide ligand was designed and placed on liposomes (d-self-peptide-labeled liposome, DSL). After mainline administration, DSL was quickly adsorbed onto hepatic phagocyte membranes (including those of Kupffer cells and liver sinusoidal endothelial cells), forming a long-lasting mask that enclosed the cell membranes and thus reducing interactions between phagocytes and subsequently injected nanoparticles. Compared with blank conventional liposomes (CL), DSL blocked the RES at a much lower dose, and the effect was sustained for a much longer time, highly prolonging the elimination half-life of the subsequently injected nanoparticles. This "don't-eat-us" strategy by DSL was further verified on the brain-targeted delivery against a cryptococcal meningitis model, providing dramatically enhanced brain accumulation of the targeted delivery system and superior therapeutic outcome of model drug Amphotericin B compared with CL. Our study demonstrates a strategy that blocks the RES by masking phagocyte surfaces to prolong nanoparticle circulation time without excess modification and illustrates its utility in enhancing nanoparticle delivery.

Oriented Assembly of Cell-Mimicking Nanoparticles via a Molecular Affinity Strategy for Targeted Drug Delivery.

Cell membrane cloaking is an emerging field in drug delivery in which specific functions of parent cells are conferred to newly formed biomimetic vehicles. A growing variety of delivery systems with diverse surface properties have been utilized for this strategy, but it is unclear whether the affinity of membrane-core pairs could guarantee effective and proper camouflaging. In this study, we propose a concise and effective "molecular affinity" strategy using the intracellular domain of transmembrane receptors as "grippers" during membrane coating. Red blood cell (RBC) membranes and cationic liposomes were adopted for fabrication, and a peptide ligand derived from the cytoplasmic protein P4.2 was prepared to specifically recognize the cytoplasmic domain of band 3, a key transmembrane receptor of erythrocytes. Once anchored onto the liposome surface, the P4.2-derived peptide would interact with the isolated RBC membrane, forming a "hidden peptide button", which ensures the right-side-out orientation. The membrane-coated liposomes exhibited an appropriate size distribution around 100 nm and high stability, with superior circulation durations compared with those of conventional PEGylated liposomes. Importantly, they possessed the ability to target Candida albicans by the interaction between the pathogenic fungus and host erythrocytes and to neutralize hemotoxin secreted by the pathogenic fungi. The curative effect of the model drug was thus substantially improved. In summary, the "molecular affinity" strategy may provide a powerful and universal approach for the construction of cell membrane-coated biomaterials and nanomedicines at both laboratory and industrial scales.

Nanoparticles Targeted against Cryptococcal Pneumonia by Interactions between Chitosan and Its Peptide Ligand.

Inspired by the fact that chitosan is a representative constituent of the ectocellular structure of Cryptococcus neoformans and a typical biomaterial for improving drug oral absorption, we designed an elegant and efficient C. neoformans-targeted drug delivery system via oral administration. A chitosan-binding peptide screened by phage display was used as the targeting moiety, followed by conjugation to the surface of poly(lactic- co-glycolic acid) nanoparticles as the drug carrier, which was then incubated with free chitosan. The noncovalently bound chitosan adheres to mucus layers and significantly enhances penetration of nanoparticles through the oral absorption barrier into circulation and then re-exposed the targeting ligand for later recognition of the fungal pathogen at the site of infection. After loading itraconazole as a model drug, our drug delivery system remarkably cleared lung infections of C. neoformans and increased survival of model mice. Currently, targeted drug delivery is mainly performed intravenously; however, the system described in our study may provide a universal means to facilitate drug targeting to specific tissues and disease sites by oral administration and may be especially powerful in the fight against increasingly severe fungal infections.

Porphyrin-grafted Lipid Microbubbles for the Enhanced Efficacy of Photodynamic Therapy in Prostate Cancer through Ultrasound-controlled In Situ Accumulation.

Photodynamic therapy (PDT) holds promise for focal therapy of prostate cancer (PCa). However, the therapeutic efficacy needs improvement, and further development of PDT for PCa has challenges, including uncertainty of photosensitizers (PSs) accumulation at the tumor site and difficulty in visualizing lesions using conventional ultrasound (US) imaging. We have developed novel porphyrin-grafted lipid (PGL) microbubbles (MBs; PGL-MBs) and propose a strategy to integrate PGL-MBs with US imaging to address these limitations and enhance PDT efficacy. METHODS: PGL-MBs have two functions: imaging guidance by contrast-enhanced ultrasound (CEUS) and targeted delivery of PSs by ultrasound targeted microbubble destruction (UTMD). PGL-MBs were prepared and characterized before and after low-frequency US (LFUS) exposure. Then, in vitro studies validated the efficacy of PDT with PGL-MBs in human prostate cancer PC3 cells. PC3-xenografted nude mice were used to validate CEUS imaging, accumulation at the tumor site, and in vivo PDT efficacy. RESULTS: PGL-MBs showed good contrast enhancement for US imaging and were converted into nanoparticles upon LFUS exposure. The resulting uniquely structured nanoparticles avoided porphyrin fluorescence quenching and efficiently accumulated at the tumor site through the sonoporation effect created with the assistance of US to achieve excellent PDT efficacy. CONCLUSIONS: This is the first preclinical investigation of MBs applied in PDT for PCa. PGL-MBs possess favorable CEUS imaging effects to enhance the localization of tumors. PGL-MBs with LFUS control PS accumulation at the tumor site to achieve highly effective PDT of PCa. This strategy carries enormous clinical potential for PCa management.

A comparative study of the antitumor efficacy of peptide-doxorubicin conjugates with different linkers.

The peptide-drug conjugates caused much attention currently. The purpose of present study was to elucidate the possible synergistic effect between ligand peptide and stimuli-responsive linkage in amphiphilic peptide-drug conjugates (APDCs) with different linkers. Especially, the superiority of each strategy as well as the synergistic effect between them was carefully investigated via the parallel comparisons of the three systems throughout of the whole study. Here, we synthesized three APDCs, namely, cRGD-SS-DOX (RSSDOX), cRGD-S-DOX (RSDOX) and cRGD-VC-DOX (RVCDOX), using doxorubicin (DOX) as a model cytotoxic agent, cRGDfC as a homing peptide, and reduction cleavable disulfide (SS), noncleavable single thioether (S) or cathepsin B cleavable valine-citrulline dipeptide (VC) as linker. The APDCs showed high drug loading capacity and they were evaluated in vitro in the integrin αvß3-overexpressing B16 cells and in vivo in tumor-bearing C57BL/6 mice. Endocytosis mechanism assay demonstrated that three types of APDCs internalized into cells through adynamin and actin depolymerizing-mediated pathway following receptor-mediated endocytosis. Notably, RSDOX or RVCDOX induced stronger antitumor efficacy, which depended on their cellular uptake levels, intracellular trafficking and the colocalization rates with lysosomes. The in vivo efficacy of RSDOX or RVCDOX was 1.4-1.7 fold of free DOX and 1.7-2.0 fold of RSSDOX, respectively. In addition, RSDOX or RVCDOX demonstrated acceptable system, tissue and blood compatibility. The compromised efficacy of RSSDOX might be due to the generation of DOX-SH during degradation of prodrug, but not DOX. Taken together, our studies suggest that certain type of APDCs can significantly decrease the toxicity of free DOX and improve therapy outcome, which provides insight for the design of peptide-drug conjugates integrating ligand peptide and stimuli-responsive linkage.

A Nanosystem of Amphiphilic Oligopeptide-Drug Conjugate Actualizing Both αvß3 Targeting and Reduction-Triggered Release for Maytansinoid.

To design a prodrug-based self-assembling nanosystem with both ligand targeting and stimuli-responsive features, and elucidate the superiority of each targeting strategy and the synergistic effect between them, we synthesized four small molecule amphiphilic peptide-drug conjugates (APDCs) using maytansinoid (DM1) as a cytotoxic agent, cRGDfK as a homing peptide, and disulfide (SS) or thioether (SMCC) as linker. Owing to their amphiphilicity, the APDCs could self-assemble into nanoparticles (APDC@NPs) which were evaluated in vitro in three different cell lines and in vivo in tumor-bearing C57BL/6 mice. The RSSD@NPs showed the strongest interaction with αvß3 integrin, highest cell uptake and intracellular free drug level, and best antitumor efficacy in vitro and in vivo, while it shared the same goodness with other test nanosystems in terms of high drug loading, EPR effect and free of potentially toxic polymers. Especially, the in vivo efficacy of RSSD@NPs was 2 fold of free DM1 which is too cytotoxic to be a drug, while the active targeted APDC@NPs demonstrated acceptable system, tissue and blood compatibility. In αvß3-positive cells or tumors, the RGD targeting contributed much more than disulfide in anticancer effect. The maximum synergism of the two strategies reached to 22 fold in vitro and 3 fold in vivo. Generally, the active targeting, prodrug and nanosystem could significantly decrease the toxicity of free DM1 and improve its therapy outcome via combining active targeting, prodrug and nanopreparation, especially the dual targeting strategies and their synergism.