Rebecca Klippstein

Polyethylene glycol conjugated polymeric nanocapsules for targeted delivery of quercetin to folate-expressing cancer cells in vitro and in vivo.

In this work we describe the formulation and characterization of chemically modified polymeric nanocapsules incorporating the anticancer drug, quercetin, for the passive and active targeting to tumors. Folic acid was conjugated to poly(lactide-co-glycolide) (PLGA) polymer to facilitate active targeting to cancer cells. Two different methods for the conjugation of PLGA to folic acid were employed utilizing polyethylene glycol (PEG) as a spacer. Characterization of the conjugates was performed using FTIR and (1)H NMR studies. The PEG and folic acid content was independent of the conjugation methodology employed. PEGylation has shown to reduce the size of the nanocapsule; moreover, zeta-potential was shown to be polymer-type dependent. Comparative studies on the cytotoxicity and cellular uptake of the different formulations by HeLa cells, in the presence and absence of excess folic acid, were carried out using MTT assay and Confocal Laser Scanning Microscopy, respectively. Both results confirmed the selective uptake and cytotoxicity of the folic acid targeted nanocapsules to the folate enriched cancer cells in a folate-dependent manner. Finally, the passive tumor accumulation and the active targeting of the nanocapsules to folate-expressing cells were confirmed upon intravenous administration in HeLa or IGROV-1 tumor-bearing mice. The developed nanocapsules provide a system for targeted delivery of a range of hydrophobic anticancer drugs in vivo.

Silver Nanoparticles: Sensing and Imaging Applications

Although silver exhibits many advantages over gold, such as higher extinction coefficients, sharper extinction bands, higher ratio of scattering to extinction, and extremely high field enhancements, it has been employed far less in the development of sensors, with the exception of sensors based on surface enhanced spectroscopies. The reason for this is the lower chemical stability of silver nanoparticles when compared to gold. Nevertheless, recent developments include means of protecting efficiently silver nanoparticles that offer far improved chemical stabilities. As a consequence, silver nanoparticles are rapidly gaining in popularity and several research groups have begun to explore alternative strategies for the development of optical sensors and imaging labels based on the extraordinary optical properties of these metal nanoparticles.

Translocation of LRP1 targeted carbon nanotubes of different diameters across the blood–brain barrier in vitro and in vivo

Brain glioblastoma and neurodegenerative diseases are still largely untreated due to the inability of most drugs to cross the blood–brain barrier (BBB). Nanoparticles have emerged as promising tools for drug delivery applications to the brain; in particular carbon nanotubes (CNTs) that have shown an intrinsic ability to cross the BBB in vitro and in vivo. Angiopep-2 (ANG), a ligand for the low-density lipoprotein receptor-related protein-1 (LRP1), has also shown promising results as a targeting ligand for brain delivery using nanoparticles (NPs). Here, we investigate the ability of ANG-targeted chemically-functionalised multi-walled carbon nanotubes (f-MWNTs) to cross the BBB in vitro and in vivo. ANG was conjugated to wide and thin f-MWNTs creating w-MWNT-ANG and t-MWNT-ANG, respectively. All f-MWNTs were radiolabelled to facilitate quantitative analyses by γ-scintigraphy. ANG conjugation to f-MWNTs enhanced BBB transport of w- and t-MWNTs-ANG compared to their non-targeted equivalents using an in vitro co-cultured BBB model consisting of primary porcine brain endothelial cells (PBEC) and primary rat astrocytes. Additionally, following intravenous administration w-MWNTs-ANG showed significantly higher whole brain uptake than the non-targeted w-MWNT in vivo reaching ~ 2% injected dose per g of brain (%ID/g) within the first hour post-injection. Furthermore, using a syngeneic glioma model, w-MWNT-ANG showed enhanced uptake in glioma brain compared to normal brain at 24 h post-injection. t-MWNTs-ANG, on the other hand, showed higher brain accumulation than w-MWNTs. However, no significant differences were observed between t-MWNT and t-MWNT-ANG indicating the importance of f-MWNTs diameter towards their brain accumulation. The inherent brain accumulation ability of f-MWNTs coupled with improved brain-targeting by ANG favours the future clinical applications of f-MWNT-ANG to deliver active therapeutics for brain glioma therapy.

Passively Targeted Curcumin-Loaded PEGylated PLGA Nanocapsules for Colon Cancer Therapy In Vivo

Clinical applications of curcumin for the treatment of cancer and other chronic diseases have been mainly hindered by its short biological half-life and poor water solubility. Nanotechnology-based drug delivery systems have the potential to enhance the efficacy of poorly soluble drugs for systemic delivery. This study proposes the use of poly(lactic-co-glycolic acid) (PLGA)-based polymeric oil-cored nanocapsules (NCs) for curcumin loading and delivery to colon cancer in mice after systemic injection. Formulations of different oil compositions are prepared and characterized for their curcumin loading, physico-chemical properties, and shelf-life stability. The results indicate that castor oil-cored PLGA-based NC achieves high drug loading efficiency (≈18% w(drug)/w(polymer)%) compared to previously reported NCs. Curcumin-loaded NCs internalize more efficiently in CT26 cells than the free drug, and exert therapeutic activity in vitro, leading to apoptosis and blocking the cell cycle. In addition, the formulated NC exhibits an extended blood circulation profile compared to the non-PEGylated NC, and accumulates in the subcutaneous CT26-tumors in mice, after systemic administration. The results are confirmed by optical and single photon emission computed tomography/computed tomography (SPECT/CT) imaging. In vivo growth delay studies are performed, and significantly smaller tumor volumes are achieved compared to empty NC injected animals. This study shows the great potential of the formulated NC for treating colon cancer.

Chemical synthesis and characterization of silver-protected vasoactive intestinal peptide nanoparticles

UNLABELLED We characterized a method to conjugate functional silver nanoparticles with vasoactive intestinal peptide (VIP), which could be used as a working model for further tailor-made applications based on VIP surface functionality. Despite sustained interest in the therapeutic applications of VIP, and the fact that its drugability could be largely improved by the attachament to functionalized metal nanoparticles, no methods have been described so far to obtain them. MATERIALS & METHODS VIP was conjugated to tiopronin-capped silver nanoparticles of a narrow size distribution, by means of proper linkers, to obtain VIP functionalized silver nanoparticles with two different VIP orientations (Ag-tiopronin-PEG-succinic-[His]VIP and Ag-tiopronin-PEG-VIP[His]). VIP intermediate nanoparticles were characterized by transmission-electron microscopy and Fourier transform infrared spectroscopy. VIP functionalized silver nanoparticles cytotoxicity was determined by lactate dehydrogenase release from mixed glial cultures prepared from cerebral cortices of 1-3 days-old C57/Bl mice. Cells were used for lipopolysaccharide stimulation at day 18-22 of culture. RESULTS Two different types of VIP-functionalized silver nanoparticles were obtained; both expose the C-terminal part of the neuropeptide, but in the first type VIP is attached to silver nanoparticle through its free amine terminus (Ag-tiopronin-PEG-succinic-[His]VIP), while in the second type, VIP N-terminus remains free (Ag-tiopronin-PEG-VIP[His]). VIP-functionalized silver nanoparticles did not compromise cellular viability and inhibited microglia-induced stimulation under inflammatory conditions. CONCLUSION The chemical synthesis procedure developed to obtain VIP-functionalized silver nanoparticles rendered functional products, in terms of biological activity. The two alternative orientations designed, reduced the constraints for chemical synthesis that depends on the nanosurface to be functionalized. Our study provides, for the first time, a proof of principle to enhance the therapeutic potential of VIP with the valuable properties of metal nanoparticles for imaging, targeting and drug delivery.

Silver Nanoparticles Interactions with the Immune System: Implications for Health and Disease

Our immune system constantly interacts with our internal environment, protects us from our external environment and provides the inherent knowledge to sense the difference between friend and foe with important implications in human health and disease (Pozo, 2008). For these reasons, it is important to identify functional alteration of key immune responses as the number of silver nano-enabled products grows while the current data strongly suggest that other related nanomaterials, such as polymer nanoparticles, fullerenes, dendrimers and gold nanoparticles, interact with the immune system.

Solvent-Free Click-Mechanochemistry for the Preparation of Cancer Cell Targeting Graphene Oxide

Polyethylene glycol-functionalized nanographene oxide (PEGylated n-GO) was synthesized from alkyne-modified n-GO, using solvent-free click-mechanochemistry, i.e., copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC). The modified n-GO was subsequently conjugated to a mucin 1 receptor immunoglobulin G antibody (anti-MUC1 IgG) via thiol–ene coupling reaction. n-GO derivatives were characterized with Fourier-transformed infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA), Bradford assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and atomic force microscopy (AFM). Cell targeting was confirmed in vitro in MDA-MB-231 cells, either expressing or lacking MUC1 receptors, using flow cytometry, confocal laser scanning microscopy (CLSM) and multiphoton (MP) fluorescence microscopy. Biocompatibility was assessed using the modified lactate dehydrongenase (mLDH) assay.

Doxorubicin enhances curcumin's cytotoxicity in human prostate cancer cells in vitro by enhancing its cellular uptake

Doxorubicin (DOX) is a widely used drug in cancer treatment. Despite its popularity, it suffers from systemic side effects and susceptibility to drug resistance. Curcumin (CURC), on the other hand, is a drug that recently gained popularity due to its wide range of biological activities, including anti-inflammatory and anti-cancer activities. Limitations to its clinical translation include its poor water solubility and the need for administration of high doses. Combinatory anti-cancer therapy has been proposed as a common approach to overcome one or more of these challenges. In this work, we propose a combinatory DOX and CURC anti-cancer therapy of prostate cancer cells in vitro. DOX and CURC were administered in the free drug and nanocapsule form, respectively. Cell size and complexity, cytotoxicity and apoptosis were studied by flow cytometry, MTT assay and sub-G1 quantification, respectively. Cellular uptake of CURC nanocapsules (CURC NCs) was quantified by fluorescence microscopy and high-performance liquid chromatography fluorescence detection. Results showed that in vitro treatment with CURC NCs in the presence of subtherapeutic concentrations of DOX, led to significant increase in prostate cancer cells (PC3) apoptosis and death. This was likely due to significantly enhanced CURC uptake by the cells. The study presents a good rationale for pursuing combinatory CURC/DOX therapy in pre-clinical tumor animal models in the near future.

Design of antibody-functionalized carbon nanotubes filled with radioactivable metals towards a targeted anticancer therapy

In the present work we have devised the synthesis of a novel promising carbon nanotube carrier for the targeted delivery of radioactivity, through a combination of endohedral and exohedral functionalization. Steam-purified single-walled carbon nanotubes (SWCNTs) have been initially filled with radioactive analogues (i.e. metal halides) and sealed by high temperature treatment, affording closed-ended CNTs with the filling material confined in the inner cavity. The external functionalization of these filled CNTs was then achieved by nitrene cycloaddition and followed by the derivatization with a monoclonal antibody (Cetuximab) targeting the epidermal growth factor receptor (EGFR), overexpressed by several cancer cells. The targeting efficiency of the so-obtained conjugate was evaluated by immunostaining with a secondary antibody and by incubation of the CNTs with EGFR positive cells (U87-EGFR+), followed by flow cytometry, confocal microscopy or elemental analyses. We demonstrated that our filled and functionalized CNTs can internalize more efficiently in EGFR positive cancer cells.

Investigating in vitro and in vivo αvβ6 integrin receptor-targeting liposomal alendronate for combinatory γδ T cell immunotherapy

Abstract The &agr;v&bgr;6 integrin receptor has been shown to be overexpressed on many types of cancer cells, resulting in a more pro‐invasive and aggressive phenotype, this makes it an attractive target for selective drug delivery. In tumours that over‐express the &agr;v&bgr;6 receptor, cellular uptake of liposomes can be enhanced using ligand‐targeted liposomes. It has previously been shown in both in vitro and in vivo studies that liposomal alendronate (L‐ALD) can sensitise cancer cells to destruction by V&ggr;9V&dgr;2 T cells. It is hypothesised that by using the &agr;v&bgr;6‐specific peptide A20FMDV2 as a targeting moiety for L‐ALD, the therapeutic efficacy of this therapy can be increased in &agr;v&bgr;6 positive tumours. Targeted liposomes (t‐L) were formulated and the targeting efficacy of targeted liposomes (t‐L) was assessed by cell uptake and cytotoxicity studies in the &agr;v&bgr;6 positive cells line A375P&bgr;6. Bio‐distribution of both L and t‐L were carried out in &agr;v&bgr;6 positive (A375P&bgr;6 and PANC0403) and &agr;v&bgr;6 negative (A375Ppuro and PANC‐1) subcutaneous tumour mouse models. Immuno‐compromised mice bearing A375P&bgr;6 experimental metastatic lung tumours were treated with L‐ALD or t‐L‐ALD as monotherapies or in combination with ex vivo‐expanded V&ggr;9V&dgr;2 T cells. In vitro, &agr;v&bgr;6‐dependant uptake of t‐L was observed, with t‐L‐ALD being more effective than L‐ALD at sensitising A375P&bgr;6 to &ggr;&dgr; T cells. Interestingly, t‐L‐ALD led to slightly higher but not significant reduction in tumour growth compared to L‐ALD, when used as monotherapy in vivo. Moreover, both L‐ALD and t‐L‐ALD led to significant reductions in tumour growth when used in combination with &ggr;&dgr; T cells in vivo but t‐L‐ALD offered no added advantage compared to L‐ALD. Graphical abstract Formulation of &agr;v&bgr;6 targeted liposomes. Liposomes were formulated with 1% DSPE‐PEG2000 Maleimide (%mol/total lipid). The peptide A20FMDV2 was modified to contain a cysteine residue at the C‐terminus. These liposomes were incubated with 25 &mgr;g peptide/&mgr;mol lipid of the peptide A20FMDV2 overnight at RT. The thiol group of the C‐terminus cysteine residue reacts with the maleimide group to form a thioether bond. Figure. No Caption available.