Untargeted Nanoparticles Research Articles

Launching triple-hit chemo attack on TNBC through nanoparticle-mediated codelivery of cisplatin-chlorambucil conjugate and venetoclax.

The BRCA1 dysfunction and HR deficiency in TNBC are responsible for high effectiveness of DNA-damaging agents in TNBC treatment. Preclinical and clinical studies confirmed the effectiveness of cisplatin in TNBC treatment. Nevertheless, the clinical utility of cisplatin is inadequate due to severe systemic side effects and resistance development. Dual-action cisplatin (IV) prodrugs provide an excellent opportunity to improve anticancer activity, reduce toxicities and minimize chance of resistance development. Therefore, in this investigation we have synthesized cisplatin-chlorambucil (CP-CBL) prodrug and loaded it with venetoclax (VTX) in phenylboronic acid conjugated TPGS-lactide nanoparticles (TNPs) to achieve tumor-targeted drug delivery thereby reducing the therapeutic dose as well as increasing the efficacy of free cisplatin, chlorambucil and venetoclax. The TNPs possessed particle size of 143nm, PDI 0.186 and entrapment efficiency of 63.5% and 56.4% for VTX and CP-CBL. The TNPs followed Higuchi release kinetic model and represented biphasic release behaviour with early burst release of drug within 2h succeeded by sustained drug release till 72h. Further, the TNPs showed∼42 folds and∼19 folds reduction in the IC50 values compared to free CP. Also, higher cellular uptake and therefore greater apoptotic index was observed for the TNPs in comparison to the untargeted nanoparticles. The TNPs further showed higher ROS generating potential, enhanced mitochondrial membrane depolarization, higher intensity of nuclear condensation and highest level of caspase-3 expression. Moreover, a noteworthy decrease in the tumor volume was noticed in the mice treated with TNPs along with lower serum toxicity biomarker levels compared to the free drugs. Overall, the developed TNPs proved to be a promising and safer strategy for inducing triple-hit action in TNBC management.

Fimbria targeting superparamagnetic iron oxide nanoparticles enhance the antimicrobial and antibiofilm activity of ciprofloxacin against quinolone-resistant E. coli.

High quinolone resistance of Escherichia coli limits the therapy options for urinary tract infection (UTI). In response to the urgent need for efficient treatment of multidrug-resistant infections, we designed a fimbriae targeting superparamagnetic iron oxide nanoparticle (SPION) delivering ciprofloxacin to ciprofloxacin-resistant E. coli. Bovine serum albumin (BSA) conjugated poly(acrylic acid) (PAA) coated SPIONs (BSA@PAA@SPION) were developed for encapsulation of ciprofloxacin and the nanoparticles were tagged with 4-aminophenyl-α-D-mannopyrannoside (mannoside, Man) to target E. coli fimbriae. Ciprofloxacin-loaded mannoside tagged nanoparticles (Cip-Man-BSA@PAA@SPION) provided high antibacterial activity (97.1 and 97.5%, respectively) with a dose of 32 μg/mL ciprofloxacin against two ciprofloxacin-resistant E. coli isolates. Furthermore, a strong biofilm inhibition (86.9% and 98.5%, respectively) was achieved in the isolates at a dose 16 and 8 times lower than the minimum biofilm eradication concentration (MBEC) of ciprofloxacin. Weaker growth inhibition was observed with untargeted nanoparticles, Cip-BSA@PAA@SPIONs, confirming that targeting E. coli fimbria with mannoside-tagged nanoparticles increases the ciprofloxacin efficiency to treat ciprofloxacin-resistant E. coli. Enhanced killing activity against ciprofloxacin-resistant E. coli planktonic cells and strong growth inhibition of their biofilms suggest that Cip-Man-BSA@PAA@SPION system might be an alternative and/or complementary therapeutic option for the treatment of quinolone-resistant E. coli infections.

Fabrication of epidermal growth factor functionalized polymeric poly(lactic-co-glycolic acid) nanoparticles loaded SN-38 and perfluorocarbon for treatment and care: investigation of antiproliferative effects and apoptosis in cervical cancer cells

Patients with cervical cancer have a significant risk of tumor recurrence and metastasis. The discovery of effective treatments for cervical cancer is urgently needed. Poly(lactic-co-glycolic acid) (PLGA) was recently discovered to offer a promising therapeutic drug carrier. Hence, we developed dual-loaded PLGA nanoparticles (NPs) as a drug carrier to combat this problem with current chemotherapeutic drugs for cervical cancer. We engineered PLGA NPs with epidermal growth factor (EGF) functionalization and co-loaded them with SN-38 and perfluorocarbon (PC) to treat cervical cancer selectively. Cell counting kit-8 test results reveal that newly fabricated NPs effectively induce cell proliferation in cervical cancer cells. Further, flow cytometry, Hoechst 33342 staining and acridine orange and propidium iodide staining were used to determine the apoptosis of SN38/PC@EGF-PLGA NPs in HeLa and CaSki cells. And the release was pH-dependent when tested in vitro. Cervical cancer cells took up targeted SN38/PC@EGF-PLGA NPs at a greater rate than untargeted NPs. Furthermore, HeLa and CaSki cells were more sensitive to apoptosis induction and cell viability suppression when exposed to SN38/PC@EGF-PLGA NPs than nontargeted NPs. The findings of this study improve the exploration of SN38/PC@EGF-PLGA NPs in the new development of effective drug candidates for highly invasive cervical cancer in future.

Can targeted nanoparticles distinguish cancer metastasis from inflammation?

Targeting ligands have been widely used to increase the intratumoral accumulation of nanoparticles and their uptake by cancer cells. However, these ligands aim at targets that are often also upregulated in inflamed tissues. Here, we assessed the ability of targeted nanoparticles to distinguish metastatic cancer from sites of inflammation. Using common targeting ligands and a 60-nm liposome as a representative nanoparticle, we generated three targeted nanoparticle (NP) variants that targeted either fibronectin, folate, or αvβ3 integrin, whose deposition was compared against that of standard untargeted NP. Using fluorescently labeled NPs and ex vivo fluorescence imaging of organs, we assessed the deposition of the NPs into the lungs of mice modeling 4 different biological landscapes, including healthy lungs, aggressive metastasis in lungs, dormant/latent metastasis in lungs, and lungs with general pulmonary inflammation. Among the four NP variants, fibronectin-targeting NP and untargeted NP exhibited the highest deposition in lungs harboring aggressive metastases. However, the deposition of all targeted NP variants in lungs with metastasis was similar to the deposition in lungs with inflammation. Only the untargeted NP was able to exhibit higher deposition in metastasis than inflammation. Moreover, flow-cytometry analysis showed all NP variants accumulated predominantly in immune cells rather than cancer cells. For example, the number of NP+ macrophages and dendritic cells was 16-fold greater than NP+ cancer cells in the case of fibronectin-targeting NP. Overall, targeted NPs were unable to distinguish cancer metastasis from general inflammation, which may have clinical implications to the nanoparticle-mediated delivery of cancer drugs.

Efficacy analysis of targeted nanodrug for non-small cell lung cancer therapy.

Biological macromolecules have been widely used as biomedical carriers in treating non-small cell lung cancer (NSCLC) due to their biocompatibility, targeting, biodegradability, and antitumor efficacy. Nanotechnology has been used in clinics to treat many diseases, including cancer. Nanoparticles (NPs) can accumulate drugs into tumors because of their enhanced permeability and retention (EPR) effects. However, the lack of active targeting ligands affects NPs drug delivery. Arginine-glycine-aspartic (RGD), as a targeting ligand, has distinct advantages in targeting and safety. In the present study, an RGD peptide-modified nanogel called RGD-polyethylene glycol-poly (L-phenylalanine-co-L-cystine) (RGD-PEG-P (LP-co-LC-P (LP-co-LC) was investigated to deliver vincristine (VCR) as NSCLC therapy. The VCR-loaded targeted nanoparticle (RGD-NP/VCR) demonstrated excellent antitumor efficacy compared to the free drug (VCR) and untargeted nanoparticle (NP/VCR) without any significant side effects. RGD-NP/VCR has better tumor inhibition and fewer side effects, indicating its potential benefit in NSCLC treatment.

An engineered three-in-one hybrid nanosystem from elastin-like polypeptides for enhanced cancer suppression

Efficiently delivering therapeutic peptides or proteins for cancer therapy remains a significant challenge. Elastin-like polypeptides (ELPs), engineered peptides inspired by natural tropoelastin, have been widely used as efficient platforms for drug delivery. Herein, we fabricated a three-in-one hybrid nanosystem based on ELPs-mCherry fusion proteins for targeting delivery of antitumor peptide (PCK3145) to colon adenocarcinoma. GE11 peptide-decorated ELPs nanoparticles could preferentially bind to the EGFR-expressed colon cancers in vitro and in vivo. GE11&PCK-ELPs-mCherry treatment could trigger a 2-fold increase in apoptotic cell death compared with untargeted nanoparticles. In mice bearing colon cancer xenograft, our multifunctional nanoparticles could significantly suppress tumor growth in vivo. Our study showed the great potential of tumor-targeted ELPs nanocarriers in delivering antitumor peptides or proteins for enhanced cancer therapy.

Molecular imaging of experimental atherosclerosis using anti-malondialdehyde-modified low-density lipoprotein humanised antibody fragment targeted nanoparticles

Abstract Introduction Oxidative modification of low-density lipoprotein (LDL), for example by malondialdehyde (MDA) adduction with subsequent uptake by macrophages to form foam cells and later the plaque necrotic core, is a key initiating event in atherogenesis. Accordingly, a larger lipid necrotic core is a key plaque vulnerability factor, predisposing plaques to rupture and subsequent thrombosis and development of an acute coronary syndrome. Thus, MDA-LDL is an attractive focus for the molecular targeting of atherosclerotic plaques. Purpose To develop antibody fragment-targeted nanoparticles that can be utilised for both the molecular imaging and therapeutics of vulnerable atherosclerotic plaques. Methods LO1 is an IgG3k natural monoclonal murine antibody that reacts with MDA-LDL. Humanised LO1Fab fragments have been engineered to reduce immunogenicity and improve lesion penetration. These humanised LO1Fab fragments were used to functionalise fluorescent poly(lactic-co-glycolic acid) (PLGA) - polyethylene glycol (PEG) nanoparticles. Nanoparticle in vitro function was assessed, prior to fluorescence molecular tomography (FMT) co-registered with micro-CT, four-hours after iv injection in atherosclerotic LDL-receptor−/− mice fed a high-fat diet for 40-weeks. Results Humanised LO1Fab fragment conjugated fluorescent PLGA-PEG nanoparticles were formulated with 210nm size and polydispersity index (variability of nanoparticle size around the average) of <0.2. Antibody conjugation efficiency was 30%. In vitro function was confirmed on ELISA versus the blank untargeted nanoparticles with MDA-LDL on solid phase, detecting nanoparticle presence via the conjugated LO1Fab, PEG corona or fluorescence. Fluorescence microscopy on stained aortic root cryosections from atherosclerotic mice confirmed binding to fatty lesions. Construct in vivo in half-life was 90-minutes for both the targeted and untargeted nanoparticles in a two-phase model in LDL-receptor−/− mice, based on fluorescence analysis of serial tail vein blood samples. There was greater uptake in the region-of-interest (heart and aortic arch vessels) in mice injected with LO1Fab-conjugated nanoparticles versus untargeted nanoparticles (mean ± standard deviation) (64.7±22.9 versus 25.2±26.5pmol of Cy5; n=7; p=0.02). Ex vivo analysis fluorescence reflectance imaging and quantitative FMT of the extracted aortae confirmed these findings (1.0±0.3 versus 0.5±0.2pmol of Cy5; n=7; p=0.002; Figure 1). Conclusions Humanised antibody Fab fragment fluorescent nanoparticles have been developed that successfully target MDA-LDL and localise to atherosclerotic plaques in murine experimental atherosclerosis. These targeted nanoparticles have the potential to amplify fluorescent signal for imaging and carry a therapeutic cargo for targeted drug delivery direct to atherosclerotic plaques. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Wellcome Trust Clinical Research Fellowship

BTN-PEG-PCL nanoparticles for targeted delivery of curcumin: In vitro and in Ovo assessment

The major challenge of curcumin (CUR) therapy is the low bioavailability in the tumor cells, which could be solved by targeting and manipulating nanoparticles (NPs) in the drug delivery systems (DDS) through enhancement of CUR uptake. In our study, ligand-targeted micelle NPs including BTN-poly ethylene glycol-polycaprolactone(PCL) (BTNcment-PEG-PCL) and mono methoxy poly (ethylene glycol)-poly (caprolactone) (mPEG-PCL) diblock co-polymers series were synthesized by way of a single-step nano-precipitation method and characterized by H NMR, FT-IR, DSC, DLS, TEM, and GPC techniques. Following CUR-loading on the NP series, cytotoxicity and cellular uptake of CUR-BTN-PEG-PCL and CUR-mPEG-PCL on human breast adenocarcinoma (MCF-7), Human embryonic kidney (HEK293), and human umbilical vein endothelial (HUVEC) cell lines were evaluated by MTT assay and microscope fluorescence, respectively. Then, in vitro studies of targeted and untargeted NPs for the cell cycle and apoptosis assessment using flow cytometry have shown that CUR in CUR-BTN-PEG-PCL as a novel and targeted nanocarrier increases the cell population of Sub G1 and apoptosis. Moreover, in Ovo chick chorioallantoic membrane (CAM) assay demonstrated that CUR-BTN-PEG-PCL has notably decreased tumor cell proliferation and angiogenesis due to enhanced CUR bioavailability for the site-specific tumor. In conclusion, BTN-PEG–PCL NPs as a novel and efficiently targeted nanocarrier predominantly improved CUR potential for cancer therapy.

Cholecystokinin-B Receptor-Targeted Nanoparticle for Imaging and Detection of Precancerous Lesions in the Pancreas.

Survival from pancreatic cancer remains extremely poor, in part because this malignancy is not diagnosed in the early stages, and precancerous pancreatic intraepithelial neoplasia (PanIN) lesions are not seen on routine radiographic imaging. Since the cholecystokinin-B receptor (CCK-BR) becomes over-expressed in PanIN lesions, it may serve as a target for early detection. We developed a biodegradable fluorescent polyplex nanoparticle (NP) that selectively targets the CCK-BR. The NP was complexed to a fluorescent oligonucleotide with Alexa Fluor 647 for far-red imaging and to an oligonucleotide conjugated to Alexa Fluor 488 for localization by immunohistochemistry. Fluorescence was detected over the pancreas of five- to ten-month-old LSL-KrasG12D/+; P48-Cre (KC) mice only after the injection of the receptor target-specific NP and not after injection of untargeted NP. Ex vivo tissue imaging and selective immunohistochemistry confirmed particle localization only to PanIN lesions in the pancreas and not in other organs, supporting the tissue specificity. A human pancreas tissue microarray demonstrated immunoreactivity for the CCK-BR only in the PanIN lesions and not in normal pancreas tissue. The long-term goal would be to develop this imaging tool for screening human subjects at high risk for pancreatic cancer to enable early cancer detection.

Polyamide Amine Dendrimer Nanoparticles Conjugated with Cis-Diamminedichloroplatinum (CDDP) and miRNA-200c Inhibitor Suppresses Lung Cancer Cell Proliferation

Our study elucidates the effect of folate polyamide amine dendrimer nanoparticles targeting delivery of miRNA-200c inhibitor and CDDP on lung cancer cells proliferation. We established polyamide amine dendrimer nanoparticles binding with CDDP and miRNA-200c inhibitor (Den-PEI-CDDP-siRNA-FA), TEM was employed to detect the morphology of nanoparticles. Agarose gel assay was selected for stabilization test. Cell proliferation were detected by trypanosoma blue exclusion method. The expression of miRNA-200c targeted APKPA12 and apoptosis-related proteins were detected by Western blot and PCR. Finally, apoptosis was analyzed by flow cytometry. Den-PEI-CDDP-siRNA-FA nanoparticles showed excellent stability and drug encapsulation ability. Nanoparticles targeting for FRA to co-deliver siRNA and CDDP could significantly promote cell apoptosis, increase apoptosis-related protein expression, and inhibit cell proliferation. Besides, nanoparticles exerted less venomous effect than untargeted nanoparticles in MRC9 lung fibroblast. Den nanoparticle targeting FRA might be used as the carrier for joint applications with siRNA and CDDP for treating lung cancer.

The dependence of radio-sensitization efficiency on mitochondrial targeting with NaGdF4:Yb,Er nanoparticles

Radio-sensitization is highly desired to reduce side-effect of the harsh dose of radiation therapy (RT), for which nanoparticles with high atomic number elements provide a promising tool. However, insufficient knowledge on utilizing the interaction between nanoparticles and cancerous cells hampers the improvement of therapeutic outcome. We herein employed NaGdF4:Yb,Er nano-crystals as the sensitizer, and modified them with a tumor targeting agent and a mitochondria targeting moiety, separately and jointly, to achieve varied extent of mitochondrial accumulation. We observed that NaGdF4:Yb,Er nano-crystal, even unmodified with targeting ligands, is effective for radio-sensitization. Furthermore, the extent of mitochondrial targeting was responsible for sensitization efficiency both in vitro and in vitro. By RNA sequencing technique, the result was ascribed to the reactive oxygen species (ROS) mediated TNF-JNK pathway and cell cycle arrest besides breaking DNA, in contrast to only DNA damage only with those untargeted nanoparticles. Our work indicated that ROS generated by the irradiation can be utilized by activating an alternative apoptotic pathway with mitochondrial targeting nanoparticles, and therefore may suggest an approach for the enhancement of radio-sensitization. Statement of significanceRadiosensitization by nanoparticles could reduce the burden of cancer due to lowering the dose of radiation therapy and reducing side-effect. How to fully utilize the interactions of irradiation-nanoparticles-biotissues remains a challenge for improving the outcome of radiosensitization. In this manuscript, by modifying tumor-targeting and mitochondria-targeting ligands on nanoparticles, separately and jointly, we demonstrated that the radiosensitization efficiency of NaGdF4:Yb,Er nanoparticle depends on the extent of accumulation near mitochondria. By RNA-seq technique, the RT sensitization with mitochondrial targeting was ascribed to ROS-mediated TNF-JNK pathway and cell cycle arrest, in contrast to only DNA breaks with untargeted nanoparticles. The results suggested a strategy for better utilization of the energy of therapeutic irradiation and demonstrate that subcellular targeting is a potent factor for designing nanoparticulate radiosensitizers.

Radiosensitization With Nanoparticles Targeting to Mitochondria

Sensitizing radiation therapy (RT) for cancer is highly desired to reduce side-effect of the harsh dose of irradiation, and nanoparticles with high atomic number elements provide a promising tool. However, insufficient knowledge on utilizing the interaction between nanoparticles and cancerous cells hampers the improvement of therapeutic outcome. We herein employed NaGdF4:Yb,Er nanocrystals as the sensitizer, and modified them with a tumor targeting agent and a mitochondria targeting moiety, separately and jointly, to achieve varied extent of mitochondrial accumulation. We observed that NaGdF4:Yb,Er nanocrystal, even unmodified with targeting ligands, is effective for radiosensitization. Furthermore, the extent of mitochondrial targeting was responsible for sensitization efficiency both in vitro and in vivo. By RNA sequencing (RNA-seq) technique, the result was ascribed to the reactive oxygen species (ROS) mediated TNF-JNK pathway and cell cycle arrest, rather than breaking DNA with those untargeted nanoparticles. Our work indicated that ROS generated by the irradiation can be utilized by activating an alternative apoptotic pathway with mitochondrial targeting nanoparticles, and therefore may suggest an approach for the enhancement of radiosensitization.

A novel MRI contrast agent for identifying hyperphosphorylative neurons as a marker of future tau pathology

AbstractBackgroundHyperphosphorylated tau (pTau) is a precursor to tau tangle formation in Alzheimer's disease (AD). We have identified aptamers that bind to specific cell membrane receptors of human neuronal cells that are hyperphosphorylating tau. In this work, we built a novel targeted nanoparticle magnetic resonance imaging (MRI) contrast agent, TauX, that binds such neurons, and tested its efficacy in vivo in the P301S mouse model of tau pathology.MethodDNA aptamers preferentially binding on the cell membrane of hyperphosphorylative SH‐SY5Y cells were selected using a modified SELEX protocol. Mass spectrometric proteomic analysis of the bound aptamers was used to identify the binding targets on the cell‐surface, and target presence was validated on both mouse and Alzheimer’s disease patient brain tissues. P301S transgenic mice (n=8) and age‐matched wild type mice (n=6) were intravenously injected TauX contrast agent and imaged by MRI using a T1‐weighted spin‐echo (T1w‐SE) sequence at 2 months of age. An additional control group of transgenic mice (n=8) received untargeted gadolinium nanoparticle contrast agent. T1‐weighted images were acquired on 1T MRI scanner at pre‐ and 4 days post‐contrast injection. Receiver operator characteristic (ROC) curves were generated with a six‐point ordinal scale to assess sensitivity and specificity for TauX. Mice were aged to 8 months and their brains were histologically assessed with AT100 antibody to confirm pTau pathology.ResultTauX‐enhanced post‐contrast images showed MR signal enhancement in most of the transgenic mice that went on to develop tau pathology 4‐6 months later, but not in wild type animals lacking tau pathology (Figure 1). Transgenic mice given untargeted nanoparticle contrast also did not show MR signal enhancement (Figure 1). AT100 staining of P301S cortical brain sections demonstrated elevated pTau levels related to wild type counterparts (Figure 2). ROC curve constructed over the entire tested group, including controls, showed overall AUC and accuracy ∼0.8 (Figure 3).ConclusionA novel MRI contrast agent that can identify mice that develop tau pathology several months in the future has been demonstrated. If successful in humans, this agent could advance the identification of patients at risk of tau pathology to a very early pre‐symptomatic stage of the disease.

Anti-MAdCAM-1-Conjugated Nanocarriers Delivering Quantum Dots Enable Specific Imaging of Inflammatory Bowel Disease.

PurposeAssessment of inflammatory bowel disease (IBD) currently relies on aspecific clinical signs of bowel inflammation. Specific imaging of the diseased bowel regions is still lacking. Here, we investigate mucosal addressin cell adhesion molecule 1 (MAdCAM-1) as a reliable and specific endothelial target for engineered nanoparticles delivering imaging agents to obtain an exact mapping of diseased bowel foci.Materials and MethodsWe generated a nanodevice composed of PLGA-PEG coupled with anti-MAdCAM-1 antibody half-chains and loaded with quantum dots (P@QD-MdC NPs). Bowel localization and systemic biodistribution of the nanoconjugate were analyzed upon injection in a murine model of chronic IBD obtained through repeated administration of dextran sulfate sodium salt. Specificity for diseased bowel regions was also assessed ex vivo in human specimens from patients with IBD. Potential for development as contrast agent in magnetic resonance imaging was assessed by preliminary study on animal model.ResultsSynthesized nanoparticles revealed good stability and monodispersity. Molecular targeting properties were analyzed in vitro in a cell culture model. Upon intravenous injection, P@QD-MdC NPs were localized in the bowel of colitic mice, with enhanced accumulation at 24 h post-injection compared to untargeted nanoparticles (p<0.05). Nanoparticles injection did not induce histologic lesions in non-target organs. Ex vivo exposure of human bowel specimens to P@QD-MdC NPs revealed specific recognition of the diseased regions vs uninvolved tracts (p<0.0001). After loading with appropriate contrast agent, the nanoparticles enabled localized contrast enhancement of bowel mucosa in the rectum of treated mice.ConclusionP@QD-MdC NPs efficiently detected bowel inflammation foci, accurately following the expression pattern of MAdCAM-1. Fine-tuning of this nanoconjugate with appropriate imaging agents offers a promising non-invasive tool for specific IBD diagnosis.

Ligand Density Controls C-Type Lectin-Like Molecule-1 Receptor-Specific Uptake of Polymer Nanoparticles.

The newest generation of drug delivery systems (DDSs) exploits ligands to mediate specific targeting of cells and/or tissues. However, studies investigating the link between ligand density and nanoparticle (NP) uptake are limited to a small number of ligand-receptor systems. C-type lectin-like molecule-1 (CLL1) is uniquely expressed on myeloid cells, which enables the development of receptors specifically targeting treat various diseases. This study aims to investigate how NPs with different CLL1 targeting peptide density impact cellular uptake. To this end, poly(styrene-alt-maleic anhydride)-b-poly(styrene) NPs are functionalized with cyclized CLL1 binding peptides (cCBP) ranging from 240 ± 12 to 31000 ± 940 peptides per NP. Unexpectedly, the percentage of cells with internalized NPs is decreased for all cCBP-NP designs regardless of ligand density compared to unmodified NPs. Internalization through CLL1 receptor-mediated processes is further investigated without confounding the effects of NP size and surface charge. Interestingly, high density cCBP-NPs (>7000 cCBP per NP) uptake is dominated by CLL1 receptor-mediated processes while low density cCBP-NPs (≈200 cCBP per NP) and untargeted NP occurred through non-specific clathrin and caveolin-mediated endocytosis. Altogether, these studies show that ligand density and uptake mechanism should be carefully investigated for specific ligand-receptor systems for the design of targeted DDSs to achieve effective drug delivery.

Dual-Targeted Delivery of Nanoparticles Encapsulating Paclitaxel and Everolimus: a Novel Strategy to Overcome Breast Cancer Receptor Heterogeneity.

The intratumoral heterogeneity observed in breast cancer (BC), in particular with regard to cell surface receptor expression, can hinder the success of many targeted cancer therapies. The development of novel therapeutic agents that target more than one receptor can overcome this inherent property of tumors and can facilitate their selective internalization in cancer cells. The goal of this study is to develop a drug combination-loaded nanoparticle (NP) formulation that is actively-targeted to HER2 and EGFR receptors on BC cells. A polymeric NP formulation was prepared which co-encapsulated a synergistic combination of the chemotherapeutic agent, paclitaxel (PTX), and the mTOR inhibitor, everolimus (EVER), and is targeted to HER2 and EGFR receptors on BC cells using antibody Fab fragments as the targeting moieties. The physicochemical characteristics of the dual-targeted formulation (Dual-NP) were evaluated, along with its cytotoxic profile (in both, monolayer and 3D BC models), as well as the degree of cellular uptake in HER2high/EGFRmod and HER2neg/EGFRlow BC cells. Dual-NPs were found to have significantly higher cytotoxicity relative to HER2 mono-targeted (T-NPs) and untargeted NPs (UT-NPs) in HER2high/EGFRmod monolayer BC cells after 72h exposure, while no significant difference was observed in HER2neg/EGFRlow cells. However, in the HER2high/EGFRmod spheroids, the cytotoxicity of Dual-NPs was comparable to that of T-NPs. This was thought to be attributed to the previously reported downregulation of EGFR in 3D in comparison to 2D BC models. Dual-NPs had significantly higher cellular uptake relative to UT-NPs and T-NPs in HER2high/EGFRmod BC cells after 24h exposure, whereas in the HER2neg/EGFRlow cells, the increase in cellular uptake of the Dual-NPs was not as high as the level achieved in the HER2high/EGFRmod cells. Blocking HER2 and EGFR significantly reduced the uptake of T-NPs and Dual-NPs in the HER2high/EGFRmod BC cells, demonstrating specific binding to both EGFR and HER2. The dual-targeting strategy developed in this study in conjunction with a potentially promising delivery vector for a synergistic combination therapy can overcome receptor heterogeneity, yielding significant improvements in the cytotoxicity and cellular uptake in BC cells.

Altered Biodistribution and Tissue Retention of Nanoparticles Targeted with P-Glycoprotein Substrates

Low molecular weight substrates of the efflux transporter, P-glycoprotein, alter the biodistribution and tissue retention of nanoparticles following intravenous administration. Of particular interest is the retention of the targeted nanoparticles in the brain. Drug delivery to the brain is hindered by the restricted transport of drugs through the blood-brain barrier (BBB). Drugs that passively diffuse across the BBB also have large volumes of distribution; therefore, alteration of their biodistribution to increase their concentration in the brain may help to enhance efficacy and reduce off-target side effects. In this work, targeted nanoparticles were used to explore a new approach to target drugs to the brain—the exploitation of the P-glycoprotein efflux pump. The retention of nanoparticles containing a strong P-glycoprotein substrate, rhodamine 6G, tethered to a PLA nanoparticle through a PEG spacer was greater than twofold relative to untargeted nanoparticles and to nanoparticles tethered to a weaker P-glycoprotein substrate, rhodamine 123. In a P-glycoprotein knockout mouse model (mdr1a (-/-)), there were no significant differences in brain accumulation between rhodamine 6G-targeted particles and controls, strongly supporting the role of P-glycoprotein. This proof of concept report shows the potential applicability of low molecular weight P-gp substrates to alter nanoparticle biodistribution. The efficacy of medicines can be improved by diverting drugs to specific tissues. Finding new ways to target medicines to diseased tissue is an active area of research across disciplines. Drug-loaded nanoparticles, delivered to tissues of interest, are one way to accomplish this goal. The work reported in this manuscript explores the possibility of using small molecules to get nanoparticles to bind to a drug efflux pump, P-glycoprotein (P-gp), that is present in various tissues in the body. P-gp functions to remove drugs from tissues, and it is usually considered a hindrance to drug targeting. The research in this paper shows that the natural function of P-gp can be used favorably to retain nanoparticles in various tissues. The data reported in this manuscript serves to establish a proof-of-concept that low molecular weight P-gp substrates can be used to alter the biodistribution of nanoparticles. Future work includes (1) understanding the targeting mechanism(s) that lead to these results, (2) identifying FDA-approved drugs that can target nanoparticles, and (3) evaluating how nanoparticle biodistribution is altered by using P-gp substrates with different binding constants.

The fate of nanoscale drug delivery systems for cartilage is influenced by osteoarthritis progression and tissue targeting strategy

Purpose: Advancing therapeutics for osteoarthritis (OA) is a pressing clinical need. Many emerging therapies act on chondrocytes, but drug delivery to cartilage has prohibitively low efficiency due to its dense, avascular extracellular matrix. Accordingly, there is significant interest in engineering targeted nanoscale delivery vehicles to facilitate retention in cartilage for localized drug release, thereby improving therapeutic impact. Here, a polymeric nanoparticle (NP) system was developed with amine functionalization for passive cartilage targeting via electrostatic interactions, and conjugation of a type 2 collagen (col2) binding peptide for active targeting. We have shown previously that cationic NPs indeed exhibit greater particle retention in healthy cartilage, likely due to electrostatic interactions with glycosaminoglycans (GAGs), but this observation is diminished in diseased tissue as GAGs are depleted. It is conceivable that the targeting approach may need to be tuned for specific diseases states or clinical applications; therefore, the purpose of this study was to directly compare targeting methods at different stages of cartilage degeneration. We hypothesize that active targeting to the col2 matrix will provide superior tissue retention relative to passive targeting via electrostatic interactions in diseased cartilage, as the tissue becomes less anionic and the porosity increases. Methods: NPs were synthesized with a poly(lactide-co-glycolide) core and polymers on the surface: polyvinyl alcohol for stabilization and/or polyallylamine (PAA) for amine decoration and surface charge modification. For passive cartilage targeting, PAA was increased to yield a cationic NP, which is designed to target the anionic cartilage matrix. For untargeted NP controls, polyallylamine concentration was reduced until the NPs were neutrally charged. For active targeting, NPs were functionalized with col2 binding peptides via conjugation. AlexaFluor dyes were conjugated to the NPs for fluorescence labelling. NP accumulation (loading) within bovine cartilage was assessed after exposing NP suspensions to the articular surface of fresh and enzymatically digested (“OA” mimic) cartilage explants for 1hr. NP release from the explants was assessed after incubation in phosphate buffered saline (PBS) at 37°C for 24hrs. Quantification of the NP loading into and release from cartilage was determined by fluorescence measurements of the tissue homogenate and surrounding PBS media, respectively, and calculated from standard curves. In vivo distribution was performed in healthy and collagenase-induced OA knees by injecting 20uL of NPs intra-articularly. Biodistribution was assessed 48hrs post injection by isolating the major knee tissues and imaging via an In Vivo Imaging System (IVIS). Results: All NP formulations were statistically the same size by dynamic light scattering (Fig 1A). Passive and active NPs were positively charged while untargeted NPs were neutral (Fig 1B). When loading NPs into cartilage, significant improvements were observed in OA tissue relative to healthy tissue in untargeted and active targeted NPs, likely because of increased pore size and access to the col2 network (Fig 1C). Passive targeted NPs demonstrated similar loading in healthy and OA tissue, likely because the increased porosity was counteracted by the weakened electrostatic gradient associated with GAG loss. After 24 hours, most of the untargeted NPs were released from the cartilage in both healthy and OA conditions (Fig 1D). In contrast, the majority of targeted NPs were retained in healthy and OA cartilage, with active targeting resulting in greater retention than passive targeting in OA tissue (Fig 1D). After intra-articular injection, whole joint distribution of both actively and passively targeted NPs was impacted by disease state, with greater cartilage accumulation in OA relative to healthy cartilage (Fig 2). Conclusions: In vitro data suggest that the tissue condition is the factor driving NP loading into cartilage, while NP retention within the cartilage is more heavily influenced by NP targeting strategy. This trend regarding NP loading was confirmed in vivo, with considerable differences in joint tissue biodistribution between healthy and OA joints. Future work will assess the NP-cartilage interactions at longer time scales for improved clinical relevance, as well as the interplay between NP retention, release of emerging OA drugs from the NP system, and therapeutic impact in vitro and in vivo. Overall, these studies highlight the importance of evaluating NP systems across degrees of disease progression. Accordingly, patient stage of disease may inform vehicle design for optimal delivery of emerging OA therapeutics.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Paclitaxel-loaded multifunctional nanoparticles for the targeted treatment of glioblastoma

Introduction: We hypothesised that the active targeting of αvβ3 integrin overexpressed in neoangiogenic blood vessels and glioblastoma (GBM) cells combined with magnetic targeting of paclitaxel- and SPIO-loaded PLGA-based nanoparticles could improve accumulation of nanoparticles in the tumour and therefore improve the treatment of GBM.Methods: PTX/SPIO PLGA nanoparticles with or without RGD-grafting were characterised. Their in vitro cellular uptake and cytotoxicity was evaluated by fluorospectroscopy and MTT assay. In vivo safety and anti-tumour efficacy of different targeting strategies were evaluated in orthotopic U87MG tumour model over multiple intravenous injections.Results: The nanoparticles of 250 nm were negatively charged. RGD targeted nanoparticles showed a specific and higher cellular uptake than untargeted nanoparticles by activated U87MG and HUVEC cells. In vitro IC50 of PTX after 48 h was ∼1 ng/mL for all the PTX-loaded nanoparticles. The median survival time of the mice treated with magnetic targeted nanoparticles was higher than the control (saline) mice or mice treated with other evaluated strategies. The 6 doses of PTX did not induce any detectable toxic effects on liver, kidney and heart when compared to Taxol.Conclusion: The magnetic targeting strategy resulted in a better therapeutic effect than the other targeting strategies (passive, active).

Mitochondria-targeting nanomedicine: An effective and potent strategy against aminoglycosides-induced ototoxicity

We report a proof-of-concept for the development of mitochondria-targeting nanoparticles (NPs) loaded with geranylgeranylacetone (GGA) to protect against a wide range of gentamicin-induced ototoxicity symptoms in a zebrafish model. The polymeric NPs were functionalized with a mitochondrial-homing peptide (d‑Arg‑Dmt‑Orn‑Phe‑NH2) and exhibited greater mitochondrial uptake and lower gentamicin uptake in hair cells via mechanotransduction (MET) channels and tuned machinery in the hair bundle than the ordinary NPs did. Blockade of MET channels rapidly reversed this effect, indicating the reversible responses of hair cells to the targeting NPs were mediated by MET channels. Pretreatment of hair cells with mitochondria-targeting GGA-loaded NPs exhibited a superior acute or chronic protective efficacy against subsequent exposure to gentamicin compared with unmodified formulations. Mitochondrial delivery regulating the death pathway of hair cells appeared to cause the therapeutic failure of untargeted NPs. Thus, peptide-directed mitochondria-targeting NPs may represent a novel therapeutic strategy for mitochondrial dysfunction-linked diseases.