Cell Uptake Studies Research Articles

Immunoglobulin G Is a Novel Substrate for the Endocytic Protein Megalin.

Therapeutic immunoglobulin G (IgG) antibodies comprise the largest class of protein therapeutics. Several factors that influence their overall disposition have been well-characterized, including target-mediated mechanics and convective flow. What remains poorly defined is the potential for non-targeted entry into various tissues or cell types by means of uptake via cell surface receptors at those sites. Megalin and cubilin are large endocytic receptors whose cooperative function plays important physiological roles at the tissues in which they are expressed. One such example is the kidney, where loss of either results in significant declines in proximal tubule protein reabsorption. Due to their diverse ligand profile and broad tissue expression, megalin and cubilin represent potential candidates for receptor-mediated uptake of IgG into various epithelia. Therefore, the objective of the current work was to determine if IgG was a novel ligand of megalin and/or cubilin. Direct binding was measured for human IgG with both megalin and the cubilin/amnionless complex. Additional work focusing on the megalin-IgG interaction was then conducted to build upon these findings. Cell uptake studies using megalin ligands for competitive inhibition or proximal tubule cells stably transduced with megalin-targeted shRNA constructs supported a role for megalin in the endocytosis of human IgG. Furthermore, a pharmacokinetic study using transgenic mice with a kidney-specific mosaic knockout of megalin demonstrated increased urinary excretion of human IgG in megalin knockout mice when compared to wild-type controls. These findings indicate that megalin is capable of binding and internalizing IgG via a high affinity interaction.

Abstract PS3-04: Breast tumor targeted fluorescence guidance for intraoperative margin assessment

Abstract BACKGROUND: A majority of the 270,000 women diagnosed with breast cancer in the United States annually are candidates for breast conserving surgery (lumpectomy), however, up to 30% of patients require reoperation due to positive margins. The ideal resolution is to make residual in-breast disease visible at the time of surgery. The goal of this research is to develop a systemically administered fluorescent molecular imaging agent that is targeted to breast tumors for intraoperative margin assessment using fluorescence guided surgery (FGS). METHODS: To identify targetable cell-surface markers for use in FGS, gene expression profiling of publicly available Affymetrix mRNA microarray data from patients with invasive ductal or invasive lobular carcinoma tumors (n=34) and surrounding normal breast tissues (n=32) was performed. Protein expression of select targets was determined by immunohistochemistry (IHC) staining of patient tissue specimens. A humanized CEACAM6 (carcinoembryonic cell adhesion molecule 6) specific antibody fragment (scFv-Fc [IgG4]) was conjugated to the near-infrared IRDye800CW fluorescent dye (LiCor) by NHS-ester linkage to create CEACAM6-800. The conjugate was characterized for in vitro specificity by immunocytochemistry (ICC) and live cell uptake studies. As proof-of-principle, the conjugate (15 µg) was intravenously injected into nude mice bearing orthotopic human MCF-7 breast cancer mammary fat pad (MFP) xenograft tumors with endogenous expression of CEACAM6. FGS was performed using the “SurgVision” clinical imaging platform (approved for clinical use in Europe) at 24 h post-injection.RESULTS: We identified 263 genes with higher mRNA expression in tumor relative to normal. From this list, we selected 9 genes for confirmation of protein expression by IHC. Of the 9 targets, only CEACAM6 had no protein expression in normal breast tissues and robust expression in ~50% of tumors. ICC staining using the CEACAM6-800 conjugate and multiple human ductal carcinoma tumor cell lines confirmed the cell surface localization of CEACAM6. In live-cell uptake studies, CEACAM6-800 accumulated on the cell surface as early as 10 min after addition to the media and was internalized after 90 min. Blocking studies demonstrated specificity. In the proof-of-principal FGS study, we observed high fluorescence in MCF-7 MFP tumors relative to background and the tumors and their residuals were completely removed in the first attempt. CONCLUSION: We have confirmed CEACAM6 protein as a cell-surface adhesion receptor target for breast cancer FGS. The CEACAM6-800, fluorescent-dye to antibody-fragment conjugate was prepared and tested in vitro for sub-cellular localization and specific uptake. In vivo studies demonstrated specific uptake into MFP tumors and FGS for tumor resection. We are currently performing studies to determine the in vivo pharmacokinetics of CEACAM6-800 uptake and clearance in tumor and normal tissues and pre-clinical post-surgery survival using our pre-clinical models. These studies will enable future translational studies. Citation Format: Narges Tafreshi, Mikalai Budzevich, Weihong Sun, Daruka Mahadevan, Marie Catherine Lee, David L Morse. Breast tumor targeted fluorescence guidance for intraoperative margin assessment [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS3-04.

Development of a tumor extracellular pH-responsive nanocarrier by terminal histidine conjugation in a star shaped poly(lactic-co-glycolic acid)

After reaching the tumor site, nanoparticles (NPs) mostly accumulate in the periphery of the tumor, as their intra-tumoral penetration is prevented due to the low perfusion, high interstitial fluid pressure, and dense matrix present in the tumor. A pH-responsive carrier can improve tumor permeation by releasing the drug quickly in the acidic tumor pH, helping its uniform tumor distribution through diffusion. In the current study, we have developed a histidine modified star-shaped PLGA (sPLGA-His) for the tumor-targeted delivery of the drug combination of docetaxel and disulfiram. The sPLGA-His NPs exhibited a rapid pH-responsive drug release behavior, with significantly increased drug release at pH 6.5 compared to pH 7.4 in 12 h. In-vitro cytotoxicity analysis showed that the pH-sensitive sPLGA-His NPs had enhanced efficacy in both 2D and 3D cell culture models. In the cell uptake study, the sPLGA-His NPs exhibited endosomal escape and uniform cellular distribution, whereas sPLGA NPs were found to be accumulated in the endosomes. In the tumor spheroid model, deep penetration was observed with the sPLGA-His NPs, while sPLGA NPs were found to be accumulated in the periphery. Using fluorescent colocalization as well as FRET analysis, increased release of the encapsulated cargo was noticed with the sPLGA-His NPs, compared to sPLGA NPs. Altogether, the sPLGA-His NPs can be used as a tumor extracellular pH-responsive nanocarrier for efficient drug delivery to the tumor.

Synthesis and tumour cell uptake studies of gadolinium(III)\u2013phosphonium complexes

The synthesis of a new series of Gd(III)-arylphosphonium complexes is described and the solution stability of selected compounds is reported. Their lipophilicity and uptake in human glial (SVG p12) and human glioblastoma multiforme (T98G) cell lines are presented. The in vitro cytotoxicity of all complexes was determined to be low at therapeutically-relevant concentrations. Selected Gd(III) complexes are potential candidates for further investigation as theranostic agents.

Cytotoxicity Effects of Curcumin Loaded on Chitosan Alginate Nanospheres on the KMBC-10 Spheroids Cell Line.

PurposeBreast cancer is one of the most lethal types of cancer in women. Curcumin showed therapeutic potential against breast cancer, but applying that by itself does not lead to the associated health benefits due to its poor bioavailability, which appears to be primarily due to poor absorption, rapid metabolism, and rapid elimination. Moreover, poor water solubility of curcumin causes accumulation of a high concentration of curcumin and so decrease its permeability to the cell. Many strategies are employed to reduce curcumin metabolism such as adjuvants and designing novel delivery systems. Therefore, in this study sodium alginate and chitosan were used to synthesize the hydrogels that are known as biocompatible, hydrophilic and low toxic drug delivery systems. Also, folic acid was used to link to chitosan in order to actively targetfolate receptors on the cells.MethodsChitosan-β-cyclodextrin-TPP-Folic acid/alginate nanoparticles were synthesized and then curcumin was loaded on them. Interaction between the constituents of the particles was characterized by FTIR spectroscopy. Morphological structures of samples were studied by FE-SEM. Release profile of curcumin was determined by dialysis membrane. The cytotoxic test was done on the Kerman male breast cancer (KMBC-10) cell line by using MTT assay. The viability of cells was detected by fluorescent staining. Gene expression was investigated by real-time PCR.ResultsThe encapsulation of curcumin into nano-particles showed an almost spherical shape and an average particle size of 155 nm. In vitro cytotoxicity investigation was indicated as dose-respond reaction against cancer breast cells after 24 h incubation. On the other hand, in vitro cell uptake study revealed active targeting of CUR-NPs into spheroids. Besides, CXCR4 expression was detected about 30-fold less than curcumin alone. The CUR-NPs inhibited proliferation and increased apoptosis in spheroid human breast cancer cells.ConclusionOur results showed the potential of NPs as an effective candidate for curcumin delivery to the target tumor spheroids that confirmed the creatable role of folate receptors.

In vitro and in vivo antitumor effects of lupeol-loaded galactosylated liposomes

Lupeol liposomes, modified with Gal-PEG-DSPE, were developed following a thin-film dispersion method. Then, the morphology, physicochemical properties, and in vitro release properties of those liposomes were investigated. The scanning electron microscopic images showed that most of the liposomes were spherical particles; they were similar in size and uniformly dispersed. Both lupeol liposomes and Gal-lupeol liposomes exhibited an average particle size of about 100 nm. The encapsulation efficiency was greater than 85%. The encapsulation efficiency of lupeol liposome and Gal-lupeol liposome, stored with 15% sucrose as glycoprotein for 6 months, was higher than 80%; although the particle size increased, they remained within 200 nm. The cell-uptake study demonstrated that the Gal-lupeol-liposome uptake efficiency was the highest in HepG2 cells. The HepG2 cells treated with the Gal-lupeol liposomes had higher apoptotic efficiency than the lupeol liposome and free lupeol. After HepG2 cells were treated with Gal-lupeol liposome, the expressions of AKT/mTOR-related proteins (p-AKT308 and p-AKT473) were also significantly reduced than the lupeol-liposome and free lupeol group. The in vivo targeting studies showed that Gal-NR-L exhibited liver-targeting effects on FVB mice. The pharmacodynamic study was performed by transfecting AKT and c-MET via the high-pressure tail vein of FVB mice. After Gal-lupeol-L administration, the liver index and liver weight of mice were less than those non-targeted group. The histopathological study showed that the lobular structure in the mice liver was clearer, the vacuoles were more obvious, and the cytoplasm was more abundant after Gal-lupeol-L administration. Also, the qRT-PCR study showed that AFP, GPC3, and EpCAM mRNA expression levels were significantly lower than those non-targeted lupeol-liposomes.

Electrochemical Characterization of Carboplatin at Unmodified Platinum Electrodes and Its Application to Drug Uptake Studies in Ovarian Cancer Cells

Epithelial ovarian cancer (EOC) has the highest mortality rate among gynecological cancers and affects patients on all continents. Primary treatments involve surgery and the administration of chemotherapeutics, such as carboplatin, which is listed by the World Health Organization as an essential medication for the treatment of human cancers. Unfortunately, due to high rates of drug resistance of EOC towards carboplatin, cancer relapse rates are devastating, and the mechanisms of chemoresistance to carboplatin have not been adequately identified. Consequently, there is a dire need for innovative strategies that are able to detect carboplatin resistance in cancer and to further our understanding of the fundamental nature of chemoresistance towards carboplatin.The presented work describes the thorough electrochemical characterization of carboplatin. We quantify the uptake of carboplatin by ovarian cancer cells by electrochemistry to better understand mechanisms of carboplatin chemoresistance. Results emerging from this research will be important for future medical research developing novel agents to combat chemoresistance.

Synthesis and Evaluation of 68Ga- and 177Lu-Labeled (R)- vs (S)-DOTAGA Prostate-Specific Membrane Antigen-Targeting Derivatives.

Prostate-specific membrane antigen (PSMA) is overexpressed in prostate cancer cells and therefore is an attractive target for prostate cancer diagnosis and radionuclide therapy. Recently, published results from clinical studies using a new PSMA-targeting PET imaging agent, [68Ga]Ga-PSMA-093 ([68Ga]Ga-HBED-CC-O-carboxymethyl-Tyr-CO-NH-Glu), support the development of this agent for the diagnosis of prostate cancer. In this study, the HBED-CC chelating group in PSMA-093 was replaced by stereoselective (R)- or (S)-DOTAGA. This chelating group serves not only for chelating 68Ga but is also amendable for complexing other radioactive metals for radionuclide therapy. The corresponding optically pure (R)- and (S)-[68Ga/177Lu]-DOTAGA derivatives, (R)-[68Ga/177Lu]-13 and (S)-[68Ga/177Lu]-13, were successfully prepared. Comparison of radiolabeling, binding affinity, cell uptake, and biodistribution between the two isomers was performed. Radiolabeling of (R)-[177Lu]Lu-13 and (S)-[177Lu]Lu-13 at 50 °C suggested that rates of complex formation were time-dependent and the formation of (S)-[177Lu]Lu-13 was distinctly faster. The rates of complex formation for the corresponding 68Ga agents were comparable between structural isomers. The natGa and natLu equivalents showed high binding PSMA affinity (IC50 = 24-111 nM), comparable to that of the parent agent, [natGa]Ga-PSMA-093 (IC50 = 34.0 nM). Results of cell uptake and biodistribution studies in PSMA-expressing PC3-PIP tumor-bearing mice appeared to show no difference between the labeled (R)- and (S)-isomers. This is the first time that a pair of [68Ga/177Lu]-(R)- and (S)-DOTAGA isomers of PSMA agents were evaluated. Results of biological studies between the isomers showed no noticeable difference; however, the distinctions on the rate of Lu complex formation should be considered in the development of new 177Lu-DOTAGA-based radionuclide therapy agents in the future.

Cyclic RGD Peptide Targeting Coated Nano Drug Co-Delivery System for Therapeutic Use in Age-Related Macular Degeneration Disease.

Vascular endothelial growth factor (VEGF) expression increased significantly in the pathogenesis of age-related macular degeneration, which induced the formation of pathological blood vessels. Dexamethasone is an exogenous anti-angiogenic drug while bevacizumab is an endogenous anti-angiogenic drug. They both have been widely used in ophthalmology. However, independent administration is not enough to completely block the development of choroidal neovascularization (CNV), and the number of eyes vitreous injections is limited. Reasonable combination of drugs may produce significantly better therapeutic effect than single drug treatment. The cyclic RGD (cRGD) peptide has a particularly high affinity with retinal pigment epithelial cells, where VEGF secretes from. In this study, we prepared nanoparticles of bevacizumab and dexamethasone with cRGD peptide as the target (aBev/cRGD-DPPNs). The particle size of the aBev/cRGD-DPPNs was 213.8 ± 1.5 nm, SEM results showed that the nano-carriers were well dispersed and spherical. The cell uptake study demonstrated the selectivity of the aBev/cRGD-DPPN to ARPE-19 with αVβ3 over expressed. The aBev/cRGD-DPPNs had a better apoptosis induction effect and an obvious inhibitory effect on migration, invasion, and capillary-like structures formation of human umbilical vein epithelial cells. The fluorescein fundus angiography study, immunohistochemistry and histopathological evaluation showed the aBev/cRGD-DPPNs greatly reduced the development of CNV on a rabbit model.

In Vitro Performance of Dutasteride-Nanostructured Lipid Carriers Coated with Lauric Acid-Chitosan Oligomer for Dermal Delivery.

Dutasteride, licensed as an oral medicine for the treatment of benign prostatic hypoplasia, has been investigated as a treatment for androgenic alopecia. In this study, the potential for dustasteride to be delivered topically in order to reduce systemic exposure, irritation of the skin, and also cytotoxicity was explored. Chitosan oligomer (CSO) was successfully synthesised with lauric acid as a coating for a dutasteride-loaded nanostructured lipid carriers (DST-NLCs) system. DST-NLCs were prepared using a combination of melt-dispersion and ultrasonication. These negatively charged NLCs (−18.0 mV) had a mean particle size of ~184 nm, which was not significantly increased (p > 0.05) when coated with lauric acid-chitosan oligomer (CSO-LA), whilst the surface charge changed to positive (+24.8 mV). The entrapment efficiency of DST-NLCs was 97%, and coated and uncoated preparations were physically stable for up to 180 days at 4–8 °C. The drug release was slower from DST-NLCs coated with CSO-LA than from uncoated NLCs, with no detectable drug permeation through full-thickness pig ear skin from either preparation. Considering the cytotoxicity, the IC50 values for the DST-NLCs, coated and uncoated with CSO-LA were greater than for dutasteride alone (p < 0.05). DST-NLCs and empty NLCs coated with CSO-LA at 25 µM increased the cell proliferation compared to the control, and no skin irritation was observed when the DST-NLC formulations were tested using EpiDerm™. The cell and skin uptake studies of coated and uncoated NLCs incorporating the fluorescent marker Coumarin-6 showed the time-dependent uptake of Coumarin-6. Overall, the findings suggest that DST-NLCs coated with CSO-LA represent a promising formulation strategy for dutasteride delivery for the treatment of androgenic alopecia, with a reduced cytotoxicity compared to that of the drug alone and lower irritancy than an ethanolic solution of dutasteride.

PEG-PLGA nanospheres loaded with nanoscintillators and photosensitizers for radiation-activated photodynamic therapy

Photodynamic Therapy (PDT) is an effective treatment modality for cancers, with Protoporphyrin IX (PPIX)-based PDT being the most widely used to treat cancers in patients. However, PDT is limited to superficial, thin (few mm in depth) lesions that can be accessed by visible wavelength light. Interstitial light-delivery strategies have been developed to treat deep-seated lesions (i.e. prostate cancer). The most promising of these are X-ray-induced scintillation nanoparticles, which have shown potential benefits for PDT of deep-seated tumors. Herein, the design and use of a new nanoscintillator-based radiation-activated PDT (radioPDT) system is investigated in the treatment of deep-seated tumors. Poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) (PEG-PLGA) nanospheres were loaded with a scintillator (LaF3:Ce3+) and photosensitizer (PPIX) to effect radioPDT. UV–Vis spectroscopy and electron microscopy studies demonstrated efficient encapsulation of nanoscintillators and PPIX (>90% efficiency) into the PEG-PLGA nanospheres. The nanoparticles were uniform in size and approximately 100 nm in diameter. They were highly stable and functional for up to 24 h under physiological conditions and demonstrated slow release kinetics. In vitro and in vivo toxicity studies showed no appreciable drug toxicity to human skin fibroblast (GM38), prostate cancer cells (PC3), and to C57/BL mice. Cell uptake studies demonstrated accumulation of the nanoparticles in the cytoplasm of PC3 cells. When activated, fluorescent resonant energy transfer (FRET) was evident via fluorescent spectroscopy and singlet oxygen yield. Determination of stability revealed that the nanoparticles were stable for up to 4 weeks. The nanoparticle production was scaled-up with no change in properties. This nanoparticle represents a unique, optimally designed therapeutic and diagnostic agent (theranostic) agent for radioPDT with characteristics capable of potentially augmenting radiotherapy for deep-seated tumors and integrating into current cancer radiotherapy.

Experimental Design Supported Liposomal Aztreonam Delivery: In Vitro Studies.

Purpose: The present study focuses on a systemic approach to develop liposomal aztreonam as a promising dosage form for inhalation therapy in the treatment of pneumonia and explores the in-vitro antimicrobial and cell uptake efficacy. Methods: Liposomes were prepared by ethanol injection method using the lipids - soya phosphatidylcholine (SP) and cholesterol (CH). A central composite design (CCD) was employed to optimize the lipid composition to evaluate the effect on vesicle size, zeta potential and entrapment efficiency of the formulation. A numerical and graphical optimization was carried out to predict the optimized blend. The optimized formulation was characterized for vesicle size, surface charge, encapsulation, surface morphology, differential scanning calorimetry (DSC), powder X Ray Diffraction (PXRD), thermogravimetric analysis (TGA), in vitro diffusion, accelerated stability studies, antimicrobial studies on Pseudomonas aeruginosa NCIM 2200 and in vitro cell uptake studies. Results: The optimized formulation was found to have a particle size of 144 nm, a surface charge of -35 mV, with satisfactory drug entrapment. The surface morphology study proved the formation of nanosized vesicles. The drug release from liposomal matrix was biphasic in nature. The solid-state study revealed the reason for good encapsulation of drug. The moisture retention capacity was found to be minimum. The anti-microbial study revealed the potential antibacterial activity of the optimized formulation over the pure drug. The formulation was found to be safe on the epithelial cells and showed a marked increase in cellular uptake of aztreonam in a lipid carrier. Conclusion: It can be concluded that the optimized liposomal aztreonam could be considered as a promising approach for the delivery of aztreonam through inhalation.

Lutein-Loaded, Biotin-Decorated Polymeric Nanoparticles Enhance Lutein Uptake in Retinal Cells.

Age related macular degeneration (AMD) is one of the leading causes of visual loss and is responsible for approximately 9% of global blindness. It is a progressive eye disorder seen in elderly people (>65 years) mainly affecting the macula. Lutein, a carotenoid, is an antioxidant, and has shown neuroprotective properties in the retina. However, lutein has poor bioavailability owing to poor aqueous solubility. Drug delivery to the posterior segment of the eye is challenging due to the blood–retina barrier. Retinal pigment epithelium (RPE) expresses the sodium-dependent multivitamin transporter (SMVT) transport system which selectively uptakes biotin by active transport. In this study, we aimed to enhance lutein uptake into retinal cells using PLGA–PEG–biotin nanoparticles. Lutein loaded polymeric nanoparticles were prepared using O/W solvent-evaporation method. Particle size and zeta potential (ZP) were determined using Malvern Zetasizer. Other characterizations included differential scanning calorimetry, FTIR, and in-vitro release studies. In-vitro uptake and cytotoxicity studies were conducted in ARPE-19 cells using flow cytometry and confocal microscopy. Lutein was successfully encapsulated into PLGA and PLGA–PEG–biotin nanoparticles (<250 nm) with uniform size distribution and high ZP. The entrapment efficiency of lutein was ≈56% and ≈75% for lutein-loaded PLGA and PLGA–PEG–biotin nanoparticles, respectively. FTIR and DSC confirmed encapsulation of lutein into nanoparticles. Cellular uptake studies in ARPE-19 cells confirmed a higher uptake of lutein with PLGA–PEG–biotin nanoparticles compared to PLGA nanoparticles and lutein alone. In vitro cytotoxicity results confirmed that the nanoparticles were safe, effective, and non-toxic. Findings from this study suggest that lutein-loaded PLGA–PEG–biotin nanoparticles can be potentially used for treatment of AMD for higher lutein uptake.

Abstract 4060: Discovery and preclinical development of KRAS-targeting siRNA therapeutic modality for the treatment of pancreatic cancer

Abstract Pancreatic cancer is the third most common cause of death from cancer in the US. The disease has a poor prognosis with a five-year survival rate under 10%. Mutations in KRAS are driving tumor growth in about 95% of patients with pancreatic cancer. Currently there are no approved drugs that target KRAS. Recently there has been progress targeting a specific mutation, KRAS G12C with small molecules in lung cancer, but this mutation accounts for only 1% of patients with Pancreatic Ductal Adenocarcinoma (PDAC). RNA interference has the potential to directly target KRAS, but delivery to pancreas has been challenging. We have developed a lipid nanoparticle formulation that is able to distribute to pancreatic tumor tissue. The proprietary lipid nanoparticle (LNP) is comprised of an ionizable, non-immunogenic, biodegradable lipid and the composition has been tuned to favor enrichment in pancreatic tumor by screening in orthotopic pancreatic tumor mouse models. A potent siRNA with IC50 in the range of 10pM targeting KRAS directly has been identified and the activity on downstream pathways and kinetics have been demonstrated. Transfection of ND-05-1192 led to more impeded cell growth in pancreatic cancer cells (PANC-1, AsPC-1, Capan-1, SW1990 and BxPC-3) than normal cells (human hepatocyte, pancreatic cells and HUVEC cells). Western blot results demonstrated effective downregulations of key proteins involved in MAPK and PI3K signaling pathways and upregulated activities of proteins involved in apoptotic signaling pathways, such as BIM, caspase 3/7 and cleaved-PARP following inhibition of KRAS protein expression. In vivo biodistribution and tumor cell uptake study results demonstrated that LNP transported ND-05-1192 siRNA molecules to the pancreas site and pancreatic orthotopic tumor tissues with the majority of tumor cells showing LNP product uptake. The application of ND-05-1192/LNP product significantly suppressed tumor growth in PANC-1 and AsPC-1 bioluminescent orthotopic pancreatic xenograft models. Inhibition efficiency revealed by tumor weight and bioluminescent response (BLR) as the treatment end points reached up to 50% in the PANC-1 orthotopic xenografts following once weekly intravenous administration. In the orthotopic AsPC-1 model, the average BLR was significantly reduced in a dose-dependent manner. Additionally, in AsPC-1 orthotopic xenografts, the survival rate of the ND-05-1192/LNP treatment group was significantly prolonged at a 4 mg/kg dose compared to the vehicle control group. In summary, we conclude that ND-05-1192/LNP is delivered to the orthotopic pancreatic tumor and its considerable uptake by tumor cells results in KRAS target downregulation, tumor growth inhibition and overall survival benefit as demonstrated in our orthotopic xenograft models. Citation Format: Zhihong (Julie) OBrien, Li Wang, Cima Cina, Bharat Majeti, James Chu, Xiaoen Wang, Robiel Baclig, James Lin, Jose Ibarra, Dhrubamitra Chatterjee, Jung-Kang Jin, Jean-Pierre Clamme, Jennifer Daniels, Hai Wang, Jens Harborth, Wenbin Ying. Discovery and preclinical development of KRAS-targeting siRNA therapeutic modality for the treatment of pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4060.

Blood-brain barrier amenable gold nanoparticles biofabrication in aged cell culture medium.

Green fabrication of nanoscale materials is highly desirable because of associated adverse effects with conventional nanomaterial biomedical applications. Moreover, the higher selective nature of the blood-brain barrier (BBB) limits the brain ailments treatment through conventional chemotherapy, thus providing room for nanotechnology-based modalities for BBB traversing. In this contribution, we have biosynthesized gold nanoparticles from the HAuCl4 solution in the aged cells culture medium. This approach is highly facile without any other chemical utilization. The cell culture medium age and cell number can tune the Au nanoparticles (AuNPs) size from 2 to several hundred nm. The 24 h MTT assay and cell uptake studies in vitro and murine models' vital organs (liver, kidney, spleen, lung, and heart) study up to 48 h demonstrated that biosynthesized AuNPs were biocompatible and BBB amenable. Interestingly, the transferrin and cell culture medium isolated proteins were found factors responsible for HAuCl4 solution biomineralization and size control. Moreover, the protein corona on biosynthesized AuNPs could help them traverse BBB both in vitro and in vivo, suggesting their potential applications for brain disease theranostics. In conclusion, the biosynthesis of AuNPs from aged cells medium is highly facile, green, and biocompatible for brain disease theranostics.

Investigation of Cytotoxicity and Cell Uptake of Cationic Beta-Cyclodextrins as Valid Tools in Nasal Delivery.

Cyclodextrin polymers have high applicability in pharmaceutical formulations due to better biocompatibility, solubility enhancement, loading capacity and controlled drug release than their parent, cyclodextrins. The cytotoxicity and cell uptake of new cationic beta-cyclodextrin monomers and polymers were evaluated as suitable materials for nasal formulations and their protective effects on cells exposed to hydrogen peroxide were studied. PC12 and CACO-2 cells were selected as the neuronal- and epithelial-type cells, respectively, to mimic the structure of respiratory and olfactory epithelia of the nasal cavity. All cationic beta-cyclodextrin polymers tested showed dose- and time-dependent toxicity; nevertheless, at 5 µM concentration and 60 min of exposure, the quaternary-ammonium-beta-cyclodextrin soluble polymer could be recognized as nontoxic. Based on these results, a fluorescently labelled quaternary-ammonium-beta-cyclodextrin monomer and polymer were selected for uptake studies in CACO-2 cells. The monomeric and polymeric beta-cyclodextrins were internalized in the cytoplasm of CACO-2 cells; the cationic monomer showed higher permeability than the hydroxypropyl-beta-cyclodextrin, employed as comparison. Therefore, these cationic beta-cyclodextrins showed potential as excipients able to improve the nasal absorption of drugs. Furthermore, amino-beta-cyclodextrin and beta-cyclodextrin soluble polymers were able to reduce oxidative damage in PC12 and CACO-2 cells and thus could be studied as bioactive carriers or potential drugs for cell protection against oxidative stress.

Mucoadhesive microspheres of ferrous sulphate – A novel approach for oral iron delivery in treating anemia

Ferrous sulphate is a widely used oral iron supplement with low bioavailability and substantial side effects. In this study ferrous sulphate has been coated with highly mucoadhesive polymers such as hydroxypropylmethyl cellulose (HP), chitosan (CS) and carbopol (CP) by spray drying technique to produce mucoadhesive polymer coated microspheres with good yield and high encapsulation efficiency. Mucoadhesive coating may allow these microspheres to get attached to the intestine and hence better absorption of ferrous sulphate may be achieved. in vitro release studies from the microspheres show that the release follows non-Fickian zero order drug release. CP and CSHP coated microspheres showed good swelling(∼1200 to 2400 %) and mucoadhesion properties (58–95 %) indicating that they can swell and get attached to the intestine for longer time period as compared to free ferrous sulphate. All the microspheres were found to be non-cytotoxic in Caco2 cell lines and fibroblast cell lines. Cell uptake studies conducted on Caco2 cell lines showed that uptake of microspheres containing ferrous sulphate has an increased and sustained release to the cell as compared to free ferrous sulphate. Though cell uptake studies showed an increase in uptake for ferrous sulphate microspheres, comparable efficacy was observed upon administering ferrous sulphate microspheres and ferrous sulphate to phenyl hydrazine induced anemic rats.

Radiosynthesis and evaluation of [18F]FMTP, a COX-2 PET ligand.

The upregulation of cyclooxygenase-2 (COX-2) is involved in neuroinflammation associated with many neurological diseases as well as cancers of the brain. Outside the brain, inflammation and COX-2 induction contribute to the pathogenesis of pain, arthritis, acute allograft rejection, and in response to infections, tumors, autoimmune disorders, and injuries. Herein, we report the radiochemical synthesis and evaluation of [18F]6-fluoro-2-(4-(methylsulfonyl)phenyl)-N-(thiophen-2-ylmethyl)pyrimidin-4-amine ([18F]FMTP), a high-affinity COX-2 inhibitor, by cell uptake and PET imaging studies. The radiochemical synthesis of [18F]FMTP was optimized using chlorine to fluorine displacement method, by reacting [18F]fluoride/K222/K2CO3 with the precursor molecule. Cellular uptake studies of [18F]FMTP was performed in COX-2 positive BxPC3 and COX-2 negative PANC-1 cell lines with unlabeled FMTP as well as celecoxib to define specific binding agents. Dynamic microPET image acquisitionwas performed in anesthetized nude mice (n = 3), lipopolysaccharide (LPS) induced neuroinflammation mice (n = 4), and phosphate-buffered saline (PBS) administered control mice (n = 4) using a Trifoil microPET/CT for a scan period of 60min. A twofold higher binding of [18F]FMTP was found in COX-2 positive BxPC3 cells compared with COX-2 negative PANC-1 cells. The radioligand did not show specific binding to COX-2 negative PANC-1 cells. MicroPET imaging in wild-type mice indicated blood-brain barrier (BBB) penetration and fast washout of [18F]FMTP in the brain, likely due to the low constitutive COX-2 expression in the normal brain. In contrast, a ~ twofold higher uptake of the radioligand was found in LPS-induced mice brain than PBS treated control mice. Specific binding to COX-2 in BxPC3 cell lines, BBB permeability, and increased brain uptake in neuroinflammation mice qualifies [18F]FMTP as a potential PET tracer for studying inflammation.

Labeling PLA-PEG nanocarriers with IR780: physical entrapment versus covalent attachment to polylactide.

Near-infrared fluorescent dyes, such as IR780, are promising theranostics, acting as photosensitizers for photodynamic therapy and in vivo tracers in image-guided diagnosis. This work compared the uptake by macrophage-like cells of IR780 either physically associated or covalently attached to poly(D,L-lactide) (PLA) formulated as polymeric nanocapsules (NC) from a blend of PLA homopolymer and PLA-PEG block copolymer. The physicochemical characterization of both NC was conducted using asymmetric flow field-flow fractionation (AF4) analysis with static and dynamic light scattering and atomic force microscopy. The interaction of IR780 with serum proteins was evidenced by AF4 with fluorescence detection and flow cytometry in cell uptake studies. The average diameters of NC were around 120 nm and zeta potentials close to -40 mV for all NC. NC uptake by cells in different media and experimental conditions shows significantly lower fluorescence intensities for IR780 covalently linked to PLA and correspondingly low quantitative uptake. Different mechanisms of internalization were evidenced depending on the IR780 type of association to NC. Serum proteins mediate IR780 interaction with cells in a dose-dependent manner. Our results show that non-covalently linked IR780 was released from NC and accumulated in macrophage cells. Oppositely, IR780 conjugated to PLA provides stable association with NC, and its fluorescence is representative of cell uptake of the nanocarrier itself. This work strongly reinforces the importance of covalent attachment of a fluorescence dye such as IR780 to the nanocarrier to study their interaction with cells in vitro and to obtain reliable tracking in image-guided therapy. Graphical abstract.

Role of Taurine Transporter in the Retinal Uptake of Vigabatrin.

Vigabatrin (VGB) is a first-line drug used for treatment of infantile spasms. On therapeutic dose, VGB accumulates in the retina causing permanent peripheral visual field constriction. The mechanism involved in retinal accumulation of VGB is ambiguous. In the present study, mechanism of VGB transport into retina was evaluated. VGB uptake into retina was studied in vitro using human adult retinal pigment epithelial (ARPE-19) cells as a model for outer blood retinal barrier. The VGB cell uptake studies demonstrated saturation kinetics with Km value of 13.1mM and uptake was significantly increased at pH7.4 and hyperosmolar conditions indicating involvement of carrier-mediated Na+-Cl--dependent transporter. In the presence of taurine transporter (TauT) substrates (taurine and GABA) and inhibitor guanidinoethyl sulfonate (GES), the uptake of VGB decreased significantly demonstrating contribution of TauT. The VGB retinal levels in rats were decreased by 1.5- and 1.3-folds on chronic administration of GES and taurine, respectively. In conclusion, this study demonstrated the TauT involvement in VGB uptake and accumulation in retina.