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Neutrophil extracellular traps (NETs) are structures consisting of decondensed chromatin with associated proteins, including histones and antimicrobial peptides, released from activated neutrophils. They are believed to be one of the body's first lines of defense against infectious agents. Despite their beneficial effect on the immune response process, some studies indicate that their excessive formation and the associated accumulation of extracellular DNA (eDNA) together with other polyelectrolytes (F-actin) plays an important role in the pathogenesis of many diseases. Thus NETs formation and removal are clinically significant. The monoclonal antibody 2C5 has strong specificity for intact nucleohistones (NS) and targets NS in NETs as we previously confirmed. Creation of a nano preparation that can specifically recognize and destroy NETs represents the aim for treatment many diseases. 2C5 antibody functionalized micelles coated with DNase I were created to achieve this aim.

The key issue in the treatment of solid tumors is the lack of efficient strategies for the targeted delivery and accumulation of therapeutic cargoes in the tumor microenvironment (TME). Targeting approaches are designed for more efficient delivery of therapeutic agents to cancer cells while minimizing drug toxicity to normal cells and off-targeting effects, while maximizing the eradication of cancer cells. The highly complicated interrelationship between the physicochemical properties of nanoparticles, and the physiological and pathological barriers that are required to cross, dictates the need for the success of targeting strategies. Dual targeting is an approach that uses both purely biological strategies and physicochemical responsive smart delivery strategies to increase the accumulation of nanoparticles within the TME and improve targeting efficiency towards cancer cells. In both approaches, either one single ligand is used for targeting a single receptor on different cells, or two different ligands for targeting two different receptors on the same or different cells. Smart delivery strategies are able to respond to triggers that are typical of specific disease sites, such as pH, certain specific enzymes, or redox conditions. These strategies are expected to lead to more precise targeting and better accumulation of nano-therapeutics. This review describes the classification and principles of dual targeting approaches and critically reviews the efficiency of dual targeting strategies, and the rationale behind the choice of ligands. We focus on new approaches for smart drug delivery in which synthetic and/or biological moieties are attached to nanoparticles by TME-specific responsive linkers and advanced camouflaged nanoparticles.

To develop immunoliposomes modified with monoclonal cancer-specific antibody (mAb) 2C5 and co-loaded with a combination of two chemotherapeutics, in order to simultaneously target bulk cancer cells using paclitaxel and cancer stem cells (CSCs) using salinomycin to prevent cancer growth and metastases. Breast cancer cells (MDA-MB-231 and/or SK-BR-3) were chosen as models for all in vitro testing. Liposomes composed of natural phospholipids co-loaded with salinomycin and paclitaxel were prepared and physically characterized. Immunoliposomes modified with mAb 2C5 coupled to polymeric conjugate were prepared and characterized for specific targeting. Wound healing assay was performed using the combination of free drugs in vitro. In vitro studies on cellular interaction and uptake were followed by holographic imaging to study cell-killing, cell-division and proliferation inhibiting effects of the formulation. Ex-vivo study on hemolysis was investigated to check possible toxicity of the formulation. Physical characterization of the liposomes showed stable nanoparticles of consistent and desirable size range (170-220 nm), zeta potential (-13 mV to – 20 mV), polydispersity indices (

Cancer is the leading cause of economic and health burden worldwide. The commonly used approaches for the treatment of cancer are chemotherapy, radiotherapy, and surgery. Chemotherapy frequently results in undesirable side effects, and cancer cells may develop resistance. Combating drug resistance is a challenging task in cancer treatment. Drug resistance may be intrinsic or acquired and can be due to genetic factors, growth factors, the increased efflux of drugs, DNA repair, and the metabolism of xenobiotics. The strategies used to combat drug resistance include the nanomedicine-based targeted delivery of drugs and genes using different nanocarriers such as gold nanoparticles, peptide-modified nanoparticles, as well as biomimetic and responsive nanoparticles that help to deliver payload at targeted tumor sites and overcome resistance. Gene therapy in combination with chemotherapy aids in this respect. siRNA and miRNA alone or in combination with chemotherapy improve therapeutic response in tumor cells. Some natural substances, such as curcumin, quercetin, tocotrienol, parthenolide, naringin, and cyclosporin-A are also helpful in combating the drug resistance of cancer cells. This manuscript summarizes the mechanism of drug resistance and nanoparticle-based strategies used to combat it.

Combination therapy has gained a lot of attention thanks to its superior activity against cancer. In the present study, we report a cRGD-targeted liposomal preparation for co-delivery of programmed cell death ligand 1 (PD-L1) small interfering RNA (siRNA) and anemoside B4 (AB4)─AB4/siP-c-L─and evaluate its anticancer efficiency in mouse models of LLC and 4T1 tumors. AB4/siP-c-L showed a particle size of (180.7 ± 7.3) nm and a ζ-potential of (32.8 ± 1.5) mV, with high drug encapsulation, pH-sensitive release properties, and good stability in serum. AB4/siP-c-L demonstrated prolonged blood circulation and increased tumor accumulation. Elevated cellular uptake was dependent on the targeting ligand cRGD. This combination induced significant tumor inhibition in LLC xenograft tumor-bearing mice by downregulating PD-L1 protein expression and modulating the immunosuppressive microenvironment. Liposomes favored the antitumor T-cell response with long-term memory, without obvious toxicity. A similar tumor growth inhibition was also demonstrated in the 4T1 tumor model. In summary, our results indicate that cRGD-modified and AB4- and PD-L1 siRNA-coloaded liposomes have potential as an antitumor preparation, and this approach may lay a foundation for the development of a new targeted drug delivery system.

Farzana Parveen,1– 3 Asadullah Madni,2 Vladimir P Torchilin,1 Mubashar Rehman,4 Talha Jamshaid,2 Nina Filipczak,1 Nadia Rai,2,5 Muhammad Muzamil Khan,2,3 Muhammad Imran Khan6 1Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, MA, 02115, USA; 2Department of Pharmaceutics, Faculty of Pharmacy, The Islamia University of Bahawalpur, Bahawalpur, Punjab, 63100, Pakistan; 3Primary and Secondary Healthcare Department, Government of Punjab, Lahore, 54000, Pakistan; 4Department of Pharmacy, Quaid-i-Azam University, Islamabad, 45320, Pakistan; 5Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Irvine, CA, 92618, USA; 6Riphah Institute of Pharmaceutical Sciences, Riphah International University Lahore Campus, Lahore, 54000, PakistanCorrespondence: Asadullah Madni, Department of Pharmaceutics, Faculty of Pharmacy, The Islamia University of Bahawalpur, Bahawalpur, Punjab, 63100, Pakistan, Tel +92 62 925 5243, Fax +92 62 925 5565, Email asadpharmacist@hotmail.comBackground: Most of the traditional nanocarriers of cancer therapeutic moieties present dose-related toxicities due to the uptake of chemotherapeutic agents in normal body cells. The severe life-threatening effects of systemic chemotherapy are well documented. Doxorubicin, DOX is the most effective antineoplastic agent but with the least specific action that is responsible for severe cardiotoxicity and myelosuppression that necessitates careful monitoring while administering. Stimuli-sensitive/intelligent drug delivery systems, specifically those utilizing temperature as an external stimulus to activate the release of encapsulated drugs, have become a subject of recent research. Thus, it would be ideal to have a nanocarrier comprising safe excipients and controllable drug release capacity to deliver the drug at a particular site to minimize unwanted and toxic effects of chemotherapeutics. We have developed a simple temperature-responsive nanocarrier based on eutectic mixture of fatty acids. This study aimed to develop, physicochemically characterize and investigate the biological safety of eutectic mixture of fatty acids as a novel construct for temperature-responsive drug release potential.Methods: We have developed phase change material, PCM, based on a series of eutectic mixtures of fatty acids due to their unique and attractive physicochemical characteristics such as safety, stability, cost-effectiveness, and ease of availability. The reversible solid-liquid phase transition of PCM is responsible to hold firm or actively release the encapsulated drug. The eutectic mixtures of fatty acids (stearic acid and myristic acid) along with liquid lipid (oleic acid) were prepared to exhibit a tunable thermoresponsive platform. Doxorubicin-loaded lipid nanocarriers were successfully developed with combined hot melt encapsulation (HME) and sonication method and characterized to achieve enhanced permeability and retention (EPR) effect-based solid tumor targeting in response to exogenous temperature stimulus. The cytotoxicity against melanoma cell lines and in vivo safety studies in albino rats was also carried out.Results: Doxorubicin-loaded lipid nanocarriers have a narrow size distribution (94.59– 219.3 nm), and a PDI (0.160– 0.479) as demonstrated by photon correlation microscopy and excellent colloidal stability (Z.P value: − 22.7 to − 32.0) was developed. Transmission electron microscopy revealed their spherical morphology and characteristics of a monodispersed system. A biphasic drug release pattern with a triggered drug release at 41°C and 43°C and a sustained drug release was observed at 37°C. The thermoresponsive cytotoxic potential was demonstrated in B16F10 cancer cell lines. Hemolysis assay and acute toxicity studies with drug-free and doxorubicin lipid nanocarrier formulations provided evidence for their non-toxic nature.Conclusion: We have successfully developed a temperature-responsive tunable platform with excellent biocompatibility and intelligent drug release potential. The formulation components being from natural sources present superior characteristics in terms of cost, compatibility with normal body cells, and adaptability to preparation methods. The reported preparation method is adapted to avoid complex chemical processes and the use of organic solvents. The lipid nanocarriers with tunable thermoresponsive characteristics are promising biocompatible drug delivery systems for improved localized delivery of chemotherapeutic agents.Graphical Abstract: Keywords: nanostructured lipid carriers, lipid nanocarriers, thermoresponsive, eutectic mixtures, fatty acids, phase change materials, doxorubicin, acute toxicity studies, hemolysis assay

Purpose: Scientific community is always searching for alternatives for cancer treatment and promoting drug repurposing is one of the ways. Rifamycin is a well-known antibiotic, that inhibits RNA synthesis and probably can be explored as cytotoxic agent. Thus, our goal was to repurpose rifamycin for breast cancer treatment using liposomes as a delivery system. Methods: Liposomes were produced by thin-film evaporation method using egg phosphatidylcholine, cholesterol and DOPE-PEG5k (70:25:5, molar ratio). Non-encapsulated drug was removed using dialysis bags and encapsulated drug was directly determined by UV spectrophotometer. Liposomes were characterized regarding size and surface charge by dynamic light scattering. Liposomes were stored at 4°C and 25°C for stability evaluation, monitoring nanoparticles parameters and drug encapsulation. Formulation biocompatibility was evaluated by hemolysis assay. Finally, cytotoxicity of rifamycin SV and liposomes was evaluated in MCF-7 and MDA-MB-231 cell lines. Results: Blank liposomes (without drug) and rifamycin-loaded liposomes showed size smaller than 100 nm and polydispersity around 0.2, with slightly negative zeta potential. Rifamycin encapsulation in liposomes was 3mg of drug/10mg of lipid films. Both formulations were stable for at least 21 days when stored at 4°C, keeping above 60% of drug encapsulated. In the concentrations tested (0.5mg/mL of drug, 2mg/mL of lipids), liposomes formulation (blank and loaded) and free rifamycin were biocompatible and non-hemolytic, since hemolysis was below 6%. Blank liposomes were not cytotoxic in tested cell lines. Free rifamycin and rifamycin-loaded liposomes demonstrated the IC50 in the same range between 41 and 46 µg/mL in 24h and 48h for both MCF-7 and MDA-MB-231. Conclusions: Rifamycin was successfully encapsulated in liposomes, leading to a stable, biocompatible, and non-hemolytic formulation. The liposomal rifamycin demonstrated strong cytotoxicity towards cancer cells similar to that of the free drug. Citation Format: Janaina Artem Ataide, Nina Filipczak, Satya Siva Kishan Yalamarty, Priscila Gava Mazzola, Vladimir P. Torchilin. Repurposing an antibiotic for breast cancer treatment using liposomes as a delivery system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1999.

Purpose: A major hurdle in chemotherapy is multidrug resistance (MDR) observed in tumors. Downregulation of efflux proteins such as P-glycoprotein (P-gp) using small interfering RNA (siRNA) can be an effective way to treat resistant tumors. In this study, monoclonal antibody 2C5 (mAb 2C5) - PEG7k-DOPE conjugates were post-inserted into the mixed dendrimer micelles containing generation 4 (G4) polyamidoamine (PAMAM)-PEG2k-DOPE and PEG5k-DOPE. The inherent amphiphilic nature of DOPE conjugates causes the copolymers to self-assemble to form a micelle and encapsulate hydrophobic chemotherapeutic drug in its core. The siRNA electrostatically binds to the cationic charges on the G4 PAMAM dendrimer. The tumor-specific mAb 2C5 on the surface of these nano-preparations resulted in improved tumor targeting. Methods: G4 PAMAM-PEG2k-DOPE was prepared by conjugating G4 PAMAM with para-nitrophenol (pNP)-PEG2k-DOPE. To prepare 2C5-PEG7k-DOPE, mAb 2C5 was mixed with micelles containing pNP-PEG7k-DOPE and PEG5k-DOPE in sodium citrate buffer at pH 5.0 and later adjusted the pH to 8.0. In vitro evaluation of the targeted and non-targeted micelles were performed in both MDA-MB-231 and SKOV-3TR resistant cell lines. Further a xenograft MDA-MB-231 in vivo mice tumor models were performed. Results: The 2C5-conjugated mixed dendrimer micelles have a uniform size distribution with a slightly positive zeta potential. They show a stable size and zeta potential for 20 days. The morphology of the 2C5 modified micelles was confirmed using TEM imaging. A higher cellular association was observed in the micelles with the 2C5 antibody targeting using fluorescence imaging (DOPE-FITC tagged micelle) in both the cell lines. At 30 minutes the 2C5 targeted mixed dendrimer micelles showed high cellular uptake and was analyzed using flow cytometry. We further evaluated the P-gp downregulation using western blot and observed a significant downregulation of P-gp levels in the targeted formulation versus the non-targeted formulation. The in vivo tumor growth inhibition study in nude NCG 572 MDA-MB-231 tumor bearing mice showed that the immuno-micelles loaded with siMDR-1 and chemotherapeutics resulted in significantly increased therapeutic efficacy compared to non-modified mixed dendrimer micelles. Conclusions: We have evaluated the 2C5 antibody-modified mixed dendrimer micelles in different cell models, in addition to the models tested in our previous studies. We have successfully established the stability of the formulation in this study and established a 20-day shelf-life. Cellular association, internalization and P-gp downregulation studies show the superiority of the 2C5 modified dendrimer micelles upon non-targeted preparations or free actives. Citation Format: Satya Siva Kishan Yalamarty, Nina Filipczak, Xiang Li, Tanvi Vinod Pathrikar, Colin Cotter, Vladimir P. Torchilin. 2C5 antibody modified dendrimer-based mixed micelles for the treatment of multidrug resistant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1997.

Background: Neutrophil extracellular traps (NETs) are composed of processed chromatin bound to granular and selected cytoplasmic proteins and released by neutrophils. NETs consist of smooth filaments composed of stacked nucleosomes. Fully hydrated NETs have a cloud-like appearance and occupy a space 10-15-fold larger than the volume of the cells they originate from. DNases are the enzymes that cleave extracellular DNA including NETs. Together with their protective role in microbial infections, NETs are involved in multiple pathological processes and represent key events in a variety of pathologies including cancer, autoimmunity, and cardiovascular disease. Sites of NETs concentration are dangerous for the host if the process of NETs formation becomes chronic or the mechanism of NETs removal does not work. NETosis has been linked to the development of periodontitis, cystic fibrosis, type 2 diabetes, COVID-19 or rheumatoid arthritis as well as cancer progression. Purpose: Thus, the destruction of NETs is of primary significance in many pathologies. In our approach, we are focusing on mimicking one of the natural mechanisms of destroying excessive NETs by delivering deoxyribonuclease I to the specific site of pathological NETs accumulation by modifying the nanoparticles using an anti-nucleosome monoclonal antibody (2C5). The antibody is specific to nucleosomes and can recognize histones in NETs. DNase I is U.S. Food and Drug Administration (FDA)-approved active component and is commonly used in therapeutic methods of modern medicine for cystic fibrosis to clear extracellular DNA fibers in the lungs and systemic lupus erythematosus. Recent findings have also shown the effectiveness of DNase I in the digestion of NETs. However, the low serum stability and fast deactivation by environmental stimuli have been considered as the limiting factors for clinical applications of DNase I, which can be overcome by its targeted specific delivery in pharmaceutical nanocarriers. Methods: In this study, we generate NETs in vitro using human neutrophils and HL-60 cells differentiated into granulocyte-like cells. We used interleukin-8, lipopolysaccharide from E.Coli (LPS), phorbol myristate acetate (PMA), and calcium ionophore A23187 (CI) to generate the NETs. We confirmed the specificity of 2C5 toward NETs by ELISA, which showed that it binds to NETs with the specificity like that for purified nucleohistone substrate. We further utilized that feature to create two delivery systems (liposomes and micelles) for DNAse I enzyme to destroy NETs, which was confirmed by staining NETs with SYTOX Green dye and followed by flow cytometric measurements and microscopic images. Conclusions: Our results suggest that 2C5 could be used to identify and visualize NETs and serve as a ligand for NET-targeted diagnostics and therapies. Also, we proved that our carrier can successfully deliver DNase to NETs to provide their degradation. Citation Format: Nina Filipczak, Xiang Li, Gaurav Rajan Saawant, Diptanshu Pardeshi, Bharat Ashok Rajmalani, Satya Siva Kishan Yalamarty, Vladimir P. Torchilin. 2C5-surface modified nanocarriers targeting neutrophil extracellular traps [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1995.

Metastasis is considered the major cause of unsuccessful cancer therapy. The metastatic development requires tumor cells to leave their initial site, circulate in the blood stream, acclimate to new cellular environments at a remote secondary site and endure there. There are several steps in metastasis, including invasion, intravasation, circulation, extravasation, premetastatic niche formation, micrometastasis and metastatic colonization. siRNA therapeutics are appreciated for their usefulness in treatment of cancer metastasis. However, siRNA therapy as a single therapy may not be a sufficient option for control of metastasis. By combining siRNA with targeting, functional agents or small-molecule drugs have shown potential effects that enhance therapeutic effectiveness. This review addresses multidrug resistance and metastasis in breast and ovarian cancers and highlights drug-delivery strategies using siRNA therapeutics.

The innovative development of nanomedicine has promised effective treatment options compared to the standard therapeutics for cancer therapy. However, the efficiency of EPR-targeted nanodrugs is not always pleasing as it is strongly prejudiced by the heterogeneity of the enhanced permeability and retention effect (EPR). Targeting the dynamics of the EPR effect and improvement of the therapeutic effects of nanotherapeutics by using EPR enhancers is a vital approach to developing cancer therapy. Inadequate data on the efficacy of EPR in humans hampers the clinical translation of cancer drugs. Molecular targeting, physical amendment, or physiological renovation of the tumor microenvironment (TME) are crucial approaches for improving the EPR effect. Advanced imaging technologies for the visualization of EPR-induced nanomedicine distribution in tumors, and the use of better animal models, are necessary to enhance the EPR effect. This review discusses strategies to enhance EPR effect-based drug delivery approaches for cancer therapy and imaging technologies for the diagnosis of EPR effects. The effort of studying the EPR effect is beneficial, as some of the advanced nanomedicine-based EPR-enhancing approaches are currently undergoing clinical trials, which may be helpful to improve EPR-induced drug delivery and translation to clinics.

Nanocarriers as drug delivery systems are promising and becoming popular, especially for cancer treatment. In addition to improving the pharmacokinetics of poorly soluble hydrophobic drugs by solubilizing them in a hydrophobic core, nanocarriers allow cancer-specific combination drug deliveries by inherent passive targeting phenomena and adoption of active targeting strategies. Nanoparticle-drug formulations can enhance the safety, pharmacokinetic profiles, and bioavailability of locally or systemically administered drugs, leading to improved therapeutic efficacy. Gene silencing by RNA interference (RNAi) is rapidly developing as a personalized field of cancer treatment. Small interfering RNAs (siRNAs) can be used to switch off specific cancer genes, in effect, "silence the gene, silence the cancer." siRNA can be used to silence specific genes that produce harmful or abnormal proteins. The activity of siRNA can be used to harness cellular machinery to destroy a corresponding sequence of mRNA that encodes a disease-causing protein. At present, the main barrier to implementing siRNA therapies in clinical practice is the lack of an effective delivery system that protects the siRNA from nuclease degradation, delivers to it to cancer cells, and releases it into the cytoplasm of targeted cancer cells, without creating adverse effects. This review provides an overview of various nanocarrier formulations in both research and clinical applications with a focus on combinations of siRNA and chemotherapeutic drug delivery systems for the treatment of multidrug resistant cancer. The use of various nanoparticles for siRNA-drug delivery, including liposomes, polymeric nanoparticles, dendrimers, inorganic nanoparticles, exosomes, and red blood cells for targeted drug delivery in cancer is discussed.

The delivery of therapeutics to brain tissues is one of the main challenges in neuropathology. For the past two decades, a variety of drug delivery systems has been designed to target components of the blood-brain barrier, including the transferrin receptor, a transmembrane glycoprotein highly expressed in the brain endothelium.In this protocol, we describe the use of transferrin protein to activate the surface of nanoparticles with the aim to direct their uptake in the brain. The molecule is bound by an amide linker to a PEGylated lipid commonly used in the preparation of lipid nanoparticles, micelles, and liposomes.

The most important goal of regenerative medicine is to repair, restore, and regenerate tissues and organs that have been damaged as a result of an injury, congenital defect or disease, as well as reversing the aging process of the body by utilizing its natural healing potential. Regenerative medicine utilizes products of cell therapy, as well as biomedical or tissue engineering, and is a huge field for development. In regenerative medicine, stem cells and growth factor are mainly used; thus, innovative drug delivery technologies are being studied for improved delivery. Drug delivery systems offer the protection of therapeutic proteins and peptides against proteolytic degradation where controlled delivery is achievable. Similarly, the delivery systems in combination with stem cells offer improvement of cell survival, differentiation, and engraftment. The present review summarizes the significance of biomaterials in tissue engineering and the importance of colloidal drug delivery systems in providing cells with a local environment that enables them to proliferate and differentiate efficiently, resulting in successful tissue regeneration.

Achievement of a high dose of drug in the tumor while minimizing its systemic side effects is one of the important features of an improved drug delivery system. Thus, developing responsive carriers for site-specific delivery of chemotherapeutic agents has become a main goal of research efforts. One of the known hallmarks of cancerous tumors is hypoxia, which offers a target for selective drug delivery. The stimuli-sensitive micellar system developed by us, (PEG-azobenzene-PEI-DOPE (PAPD) has proven to be effective in vitro. The proposed construct developed, PAPD, contains an azobenzene group as a hypoxia-sensitive moiety that triggers the shedding of the PEG layer from the nanoparticle surface under conditions of hypoxia to improve cellular uptake. Using microfluidics, we show significantly improved cellular association and penetration under hypoxia in both single cells and in a 3D tumor model. Employing an in vivo model, we demonstrate slower tumor growth that did not induce systemic side effects, including weight loss in an experimental animal model, when compared to the free drug treatment. This complex-in-nature but simple-in-design system for the simultaneous delivery of siRNA to silence the P-glycoprotein and doxorubicin with active tumor targeting and proven therapeutic efficacy represents a universal platform for the delivery of other hydrophobic chemotherapeutic agents and siRNA molecules which can be further modified.

Antibody-drug conjugate-based therapy for treatment of cancer has attracted much attention because of its enhanced efficacy against numerous cancer types. Commonly, an ADC includes a mAb linked to a therapeutic payload. Antibody, linker and payload are the three main components of ADCs. The high specificity of antibodies is integrated with the strong potency of payloads in ADCs. ADCs with potential cytotoxic small molecules as payloads, generate antibody-mediated cancer therapy. Recently, ADCs with DNA-damaging agents have shown favor over microtubule-targeting agents as payloads. Although ADC resistance can be a barrier to effectiveness, several ADC therapies have been either approved or are in clinical trials for cancer treatment. The ADC-based treatments of breast cancers, particularly TNBC, MDR and metastatic breast cancers, have shown promise in recent years. This review discusses ADC drug designs, and developed for different types of breast cancer including TNBC, MDR and metastatic breast cancer.

Dendrimers comprise a specific group of macromolecules, which combine structural properties of both single molecules and long expanded polymers. The three-dimensional form of dendrimers and the extensive possibilities for use of additional substrates for their construction creates a multivalent potential and a wide possibility for medical, diagnostic and environmental purposes. Depending on their composition and structure, dendrimers have been of interest in many fields of science, ranging from chemistry, biotechnology to biochemical applications. These compounds have found wide application from the production of catalysts for their use as antibacterial, antifungal and antiviral agents. Of particular interest are peptide dendrimers as a medium for transport of therapeutic substances: synthetic vaccines against parasites, bacteria and viruses, contrast agents used in MRI, antibodies and genetic material. This review focuses on the description of the current classes of dendrimers, the methodology for their synthesis and briefly drawbacks of their properties and their use as potential therapies against infectious diseases.

Introduction: Nano formulations continue to be a very attractive venue for the development of “smart” drug delivery systems. Emerging green (organic) formulations do not require harsh chemical solvents and are easy to prepare. Solid Core Lipid Formulations: The lipid phase (l.p.) contained a solid fatty acid (lauric acid) and liquid lipid (oleic acid) with or without Doxorubicin. 55° surfactant containing an emulsifying agent (Brij-58) mixed with nonionic surfactant (Span 80) was dropwise added. The emulsion was cooled to form temperature-activated nanoparticles (TAN). Before usage, the emulsion was activated by heating to 41°C. Experiments: B16F10 melanoma, U87-MR glioblastoma, MDA-MB-231 and MCF-7 breast cancer cell lines were analyzed on a variety of quantitative Imaging platforms. Results iCyte (CompuCyte): Laser based instrument for obtaining quantitative fluorescence and light scatter measurements. We obtained single endpoint dosage response curves to compare the TAN with free Dox, and found the TANs were effective at 4-6 times lower equivalent Dox concentrations. Holomonitor (Phase Holographic Imaging): The HM4 resides in tissue culture incubators and allows long-term medium resolution holographic imaging. It offersa complete image processing library including routines for proliferation and cell tracking. We devised methods for 4-dimensional imaging (X pos. vs. Y pos. vs Time, vs. Density). Thus, we are able to monitor the temporal effects of the compounds. Cell Explorer (Nanolive): The Cell Explorer has a a rotating mirror that directs the laser in an orbit around the sample to obtain super-resolution 3D tomographs. It enables long-term time-lapse imaging via stage-top environmental chamber. It is well suited for quantifying sub cellular components such as mitochondria and lipids. HT-2 (Tomocube): The HT-2 is a label-free ultra-high resolution holotomography system complemented by fluorescence capabilities. Combined RI and fluorescence images give the highest information content of any of the platforms. Discussion: In 2012, Dixon et al described ferroptosis as a distinct new form of cell death through the iron dependent accumulation of oxidatively damaged phospholipids. They state that a diagnostic feature is mitochondrial shrinkage and collapse. We confirmed this in our tomographic imaging images. Other features that were detected and consistent with ferroptosis are loss of nuclear contents, a thickening of the nuclear membrane, and peri-nuclear mitochondrial accumulation. In 48-hour HM4 plots control cells show numerous mitotic events. Dox treated cells show cellular enlargement but no mitotic events. In the TAN population at about 20 hours, there is sudden death occurring in the entire population. Conclusion: Our experiments suggest a high potency of the newly developed TANs. Ferroptosis is the most likely mechanism of action. Citation Format: Farzana Parveen, Nina Filipczak, Asadullah Madni, Vladimir P. Torchilin, Ed Luther. Characterization of a green, temperature activated nano formulation that drives the mechanism of doxorubicin toxicity from apoptosis to ferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5327.

Biomarkers are emerging as potentially important diagnostic tools for cancer and many other diseases. However, many current detection systems for suffer from insufficient sensitivity. To address this concern, we developed a highly sensitive biosensor, featuring monoclonal antibody-coated polystyrene nanobeads assembled in the trenches of a microchip, for the detection of cancer biomarkers. These biosensors detected nucleosomes and carcinoembryonic antigen in serum at concentrations of 62.5 and 15.6 pg/mL, respectively. Very low detection limits that suggest such devices might be beneficial for the early detection of tumors and for monitoring of patients in remission.

Dendritic cells serve as the main immune cells that trigger the immune response. We developed a simple and cost-effective nanovaccine platform based on the α1',2-mannobiose derivative for dendritic cell targeting. In previous work, we have formulated the α1,2-mannobiose-based nanovaccine platform with plasmid DNA and tested it in cattle against BoHV-1 infection. There, we have shown that the dendritic cell targeting using this nanovaccine platform in vivo can boost the immunogenicity, resulting in a long-lasting immunity. In this work, we aim to characterize the α1',2-mannobiose derivative, which is key in the nanovaccine platform. This DC-targeting strategy takes advantage of the specific receptor known as DC-SIGN and exploits its capacity to bind α1,2-mannobiose that is present at terminal ends of oligosaccharides in certain viruses, bacteria, and other pathogens. The oxidative conjugation of α1',2-mannobiose to NH2-PEG2kDa-DSPE allowed us to preserve the chemical structure of the non-reducing mannose of the disaccharide and the OH groups and the stereochemistry of all carbons of the reducing mannose involved in the binding to DC-SIGN. Here, we show specific targeting to DC-SIGN of decorated micelles incubated with the Raji/DC-SIGN cell line and uptake of targeted liposomes that took place in human, bovine, mouse, and teleost fish DCs in vitro, by flow cytometry. Specific targeting was found in all cultures, demonstrating a species-non-specific avidity for this ligand, which opens up the possibility of using this nanoplatform to develop new vaccines for various species, including humans.

The delivery of therapeutics to brain tissues is one of the main challenges in neuropathology. For the past two decades, a variety of drug delivery systems has been designed to target components of the blood-brain barrier, including the transferrin receptor, a transmembrane glycoprotein highly expressed in the brain endothelium.In this protocol, we describe the use of transferrin protein to activate the surface of nanoparticles with the aim to direct their uptake in the brain. The molecule is bound by an amide linker to a PEGylated lipid commonly used in the preparation of lipid nanoparticles, micelles, and liposomes.

New technologies in the field of vaccinology arise as a necessity for the treatment and control of many diseases. Whole virus inactivated vaccines and modified live virus ones used against Bovine Herpesvirus-1 (BoHV-1) infection have several disadvantages. Previous works on DNA vaccines against BoHV-1 have demonstrated the capability to induce humoral and cellular immune responses. Nevertheless, ‘naked’ DNA induces low immunogenic response. Thus, loading of antigen encoding DNA sequences in liposomal formulations targeting dendritic cell receptors could be a promising strategy to better activate these antigen-presenting cells (APC). In this work, a DNA-based vaccine encoding the truncated version of BoHV-1 glycoprotein D (pCIgD) was evaluated alone and encapsulated in a liposomal formulation containing LPS and decorated with MANα1-2MAN-PEG-DOPE (pCIgD-Man-L). The vaccinations were performed in mice and bovines. The results showed that the use of pCIgD-Man-L enhanced the immune response in both animal models. For humoral immunity, significant differences were achieved when total antibody titres and isotypes were assayed in sera. Regarding cellular immunity, a significant increase in the proliferative response against BoHV-1 was detected in animals vaccinated with pCIgD-Man-L when compared to the response induced in animals vaccinated with pCIgD. In addition, upregulation of CD40 molecules on the surface of bovine dendritic cells (DCs) was observed when cells were stimulated and activated with the vaccine formulations. When viral challenge was performed, bovines vaccinated with MANα1-2MAN-PEG-DOPE elicited better protection which was evidenced by a lower viral excretion. These results demonstrate that the dendritic cell targeting using MANα1-2MAN decorated liposomes can boost the immunogenicity resulting in a long-lasting immunity. Liposomes decorated with MANα1-2MAN-PEG-DOPE were tested for the first time as a DNA vaccine nanovehicle in cattle as a preventive treatment against BoHV-1. These results open new perspectives for the design of vaccines for the control of bovine rhinotracheitis. Fil: Kornuta, Claudia Alejandra. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Virología E Innovaciones Tecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Instituto de Virología E Innovaciones Tecnológicas; Argentina Fil: Bidart, Juan Esteban. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Virología E Innovaciones Tecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Instituto de Virología E Innovaciones Tecnológicas; Argentina Fil: Soria, Ivana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Virología E Innovaciones Tecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Instituto de Virología E Innovaciones Tecnológicas; Argentina Fil: Gammella, Mariela Vanesa. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Virología E Innovaciones Tecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Instituto de Virología E Innovaciones Tecnológicas; Argentina Fil: Quattrocchi, Valeria. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Virología E Innovaciones Tecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Instituto de Virología E Innovaciones Tecnológicas; Argentina Fil: Pappalardo, Juan Sebastian. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche. Instituto de Investigaciones Forestales y Agropecuarias Bariloche. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones Forestales y Agropecuarias Bariloche; Argentina Fil: Salmaso, Stefano. Università di Padova; Italia Fil: Torchilin, Vladimir P. Northeastern University; Estados Unidos Fil: Cheuquepán Valenzuela, Felipe Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina Fil: Hecker, Yanina Paola. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina Fil: Moore, Dadin Prando. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina Fil: Zamorano, Patricia Ines. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Virología E Innovaciones Tecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Instituto de Virología E Innovaciones Tecnológicas; Argentina Fil: Langellotti, Cecilia Ana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Virología E Innovaciones Tecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Instituto de Virología E Innovaciones Tecnológicas; Argentina