EGFR Targeting of Liposomal Doxorubicin Improves Recognition and Suppression of Non-Small Cell Lung Cancer.

Introduction: Despite improvements in chemotherapy and molecularly targeted therapies, the life expectancy of patients with advanced non-small cell lung cancer (NSCLC) remains less than 1 year. There is thus a major global need to advance new treatment strategies that are more effective for NSCLC. Drug delivery using liposomal particles has shown success at improving the biodistribution and bioavailability of chemotherapy. Nevertheless, liposomal drugs lack selectivity for the cancer cells and have a limited ability to penetrate the tumor site, which severely limits their therapeutic potential. Epidermal growth factor receptor (EGFR) is overexpressed in NSCLC tumors in about 80% of patients, thus representing a promising NSCLC-specific target for redirecting liposome-embedded chemotherapy to the tumor site. Methods: Herein, we investigated the targeting of PEGylated liposomal doxorubicin (Caelyx), a powerful off-the-shelf antitumoral liposomal drug, to EGFR as a therapeutic strategy to improve the specific delivery and intratumoral accumulation of chemotherapy in NSCLC. EGFR-targeting of Caelyx was enabled through its complexing with a polyethylene glycol (PEG)/EGFR bispecific antibody fragment. Tumor targeting and therapeutic potency of our treatment approach were investigated in vitro using a panel of NSCLC cell lines and 3D tumoroid models, and in vivo in a cell line-derived tumor xenograft model. Results: Combining Caelyx with our bispecific antibody generated uniform EGFR-targeted particles with improved binding and cytotoxic efficacy toward NSCLC cells. Effects were exclusive to cancer cells expressing EGFR, and increments in efficacy positively correlated with EGFR density on the cancer cell surface. The approach demonstrated increased penetration within 3D spheroids and was effective at targeting and suppressing the growth of NSCLC tumors in vivo while reducing drug delivery to the heart. Conclusion: EGFR targeting represents a successful approach to enhance the selectivity and therapeutic potency of liposomal chemotherapy toward NSCLC.

Spatio-temporal analysis of nanoparticles in live tumor spheroids impacted by cell origin and density.

Nanoparticles hold great preclinical promise in cancer therapy but continue to suffer attrition through clinical trials. Advanced, three dimensional (3D) cellular models such as tumor spheroids can recapitulate elements of the tumor environment and are considered the superior model to evaluate nanoparticle designs. However, there is an important need to better understand nanoparticle penetration kinetics and determine how different cell characteristics may influence this nanoparticle uptake. A key challenge with current approaches for measuring nanoparticle accumulation in spheroids is that they are often static, losing spatial and temporal information which may be necessary for effective nanoparticle evaluation in 3D cell models. To overcome this challenge, we developed an analysis platform, termed the Determination of Nanoparticle Uptake in Tumor Spheroids (DONUTS), which retains spatial and temporal information during quantification, enabling evaluation of nanoparticle uptake in 3D tumor spheroids. Outperforming linear profiling methods, DONUTS was able to measure silica nanoparticle uptake to 10 µm accuracy in both isotropic and irregularly shaped cancer cell spheroids. This was then extended to determine penetration kinetics, first by a forward-in-time, center-in-space model, and then by mathematical modelling, which enabled the direct evaluation of nanoparticle penetration kinetics in different spheroid models. Nanoparticle uptake was shown to inversely relate to particle size and varied depending on the cell type, cell stiffness and density of the spheroid model. The automated analysis method we have developed can be applied to live spheroids in situ, for the advanced evaluation of nanoparticles as delivery agents in cancer therapy.

Facile synthesis of lactoferrin conjugated ultra small large pore silica nanoparticles for the treatment of glioblastoma.

The blood brain barrier (BBB) and blood tumour barrier (BTB) remain a major roadblock for delivering therapies to treat brain cancer. Amongst brain cancers, glioblastoma (GBM) is notoriously difficult to treat due to the challenge of delivering chemotherapeutic drugs across the BBB and into the tumour microenvironment. Consequently, GBM has high rates of tumour recurrence. Currently, limited numbers of chemotherapies are available that can cross the BBB to treat GBM. Nanomedicine is an attractive solution for treating GBM as it can augment drug penetration across the BBB and into the heterogeneous tumour site. However, very few nanomedicines exist that can easily overcome both the BBB and BTB owing to difficulty in synthesizing nanoparticles that meet the small size and surface functionality restrictions. In this study, we have developed for the first-time, a room temperature protocol to synthesise ultra-small size with large pore silica nanoparticles (USLP, size ∼30 nm, pore size >7 nm) with the ability to load high concentrations of chemotherapeutic drugs and conjugate a targeting moiety to their surface. The nanoparticles were conjugated with lactoferrin (>80 kDa), whose receptors are overexpressed by both the BBB and GBM, to achieve additional active targeting. Lactoferrin conjugated USLP (USLP-Lf) were loaded with doxorubicin - a chemotherapy agent that is known to be highly effective against GBM in vitro but cannot permeate the BBB. USLP-Lf were able to selectively permeate the BBB in vitro, and were effectively taken up by glioblastoma U87 cells. When compared to the uncoated USLP-NPs, the coating with lactoferrin significantly improved penetration of USLP into U87 tumour spheroids (after 12 hours at 100 µm distance, RFU value 19.58 vs. 49.16 respectively). Moreover, this USLP-Lf based delivery platform improved the efficacy of doxorubicin-mediated apoptosis of GBM cells in both 2D and 3D models. Collectively, our new nano-platform has the potential to overcome both the BBB and BTB to treat GBM more effectively.

Doxorubicin-Loaded Gold Nanoarchitectures as a Therapeutic Strategy against Diffuse Intrinsic Pontine Glioma.

Diffuse Intrinsic Pontine Gliomas (DIPGs) are highly aggressive paediatric brain tumours. Currently, irradiation is the only standard treatment, but is palliative in nature and most patients die within 12 months of diagnosis. Novel therapeutic approaches are urgently needed for the treatment of this devastating disease. We have developed non-persistent gold nano-architectures (NAs) functionalised with human serum albumin (HSA) for the delivery of doxorubicin. Doxorubicin has been previously reported to be cytotoxic in DIPG cells. In this study, we have preclinically evaluated the cytotoxic efficacy of doxorubicin delivered through gold nanoarchitectures (NAs-HSA-Dox). We found that DIPG neurospheres were equally sensitive to doxorubicin and doxorubicin-loaded NAs. Colony formation assays demonstrated greater potency of NAs-HSA-Dox on colony formation compared to doxorubicin. Western blot analysis indicated increased apoptotic markers cleaved Parp, cleaved caspase 3 and phosphorylated H2AX in NAs-HSA-Dox treated DIPG neurospheres. Live cell content and confocal imaging demonstrated significantly higher uptake of NAs-HSA-Dox into DIPG neurospheres compared to doxorubicin alone. Despite the potency of the NAs in vitro, treatment of an orthotopic model of DIPG showed no antitumour effect. This disparate outcome may be due to the integrity of the blood-brain barrier and highlights the need to develop therapies to enhance penetration of drugs into DIPG.

Frontiers in the treatment of glioblastoma: Past, present and emerging.

Glioblastoma (GBM) is one of the most aggressive cancers of the brain. Despite extensive research over the last several decades, the survival rates for GBM have not improved and prognosis remains poor. To date, only a few therapies are approved for the treatment of GBM with the main reasons being: 1) significant tumour heterogeneity which promotes the selection of resistant subpopulations 2) GBM induced immunosuppression and 3) fortified location of the tumour in the brain which hinders the delivery of therapeutics. Existing therapies for GBM such as radiotherapy, surgery and chemotherapy have been unable to reach the clinical efficacy necessary to prolong patient survival more than a few months. This comprehensive review evaluates the current and emerging therapies including those in clinical trials that may potentially improve both targeted delivery of therapeutics directly to the tumour site and the development of agents that may specifically target GBM. Particular focus has also been given to emerging delivery technologies such as focused ultrasound, cellular delivery systems nanomedicines and immunotherapy. Finally, we discuss the importance of developing novel materials for improved delivery efficacy of nanoparticles and therapeutics to reduce the suffering of GBM patients.

Monitoring the heterogeneity in single cell responses to drugs using electrochemical impedance and electrochemical noise.

Impedance spectroscopy is a widely used technique for monitoring cell-surface interactions and morphological changes, typically based on averaged signals from thousands of cells. However, acquiring impedance data at the single cell level, can potentially reveal cell-to-cell heterogeneity for example in response to chemotherapeutic agents such as doxorubicin. Here, we present a generic platform where light is used to define and localize the electroactive area, thus enabling the impedance measurements for selected single cells. We firstly tested the platform to assess phenotypic changes in breast cancer cells, at the single cell level, using the change in the cell impedance. We next show that changes in electrochemical noise reflects instantaneous responses of the cells to drugs, prior to any phenotypical changes. We used doxorubicin and monensin as model drugs and found that both drug influx and efflux events affect the impedance noise signals. Finally, we show how the electrochemical noise signal can be combined with fluorescence microscopy, to show that the noise provides information on cell susceptibility and resistance to drugs at the single cell level. Together the combination of electrochemical impedance and electrochemical noise with fluorescence microscopy provides a unique approach to understanding the heterogeneity in the response of single cells to stimuli where there is not phenotypic change.

Intra-articular Treatment of Osteoarthritis with Diclofenac-Conjugated Polymer Reduces Inflammation and Pain.

The most common treatment for osteoarthritis is daily oral administration of a nonsteroidal anti-inflammatory drug such as diclofenac. This daily dosage regime is often associated with severe side effects. In this study, we explored the potential of utilizing a high molecular weight cross-linked polyurethane polymer covalently linked to diclofenac (C-DCF-PU) for intra-articular administration. We aim to exploit the advantages of local drug delivery by developing an implant with improved efficacy and reduced side effects. The polymer was synthesized from a diclofenac-functionalized monomer unit in a simple one-pot reaction, followed by cross-linking. In vitro drug release studies showed zero-order drug release for 4 days, followed by a gradual decline in drug release rate until diclofenac was depleted after 15 days. The cross-linked polymer was triturated to yield an injectable microgel formulation for administration. Whole animal fluorescence imaging of the rhodamine-labeled C-DCF-RH-PU showed good retention of the polymer in the knee joints of healthy rats, with approximately 30% of the injected dose still present 2 weeks post intra-articular administration. In a reactivation arthritis animal model, the C-DCF-RH-PU formulation reduced pain and significantly reduced inflammation after a short lag phase, showing that this drug delivery system warrants further development for long-term treatment of osteoarthritis with the benefit of reduced side effects.

Biologically Targeted Magnetic Hyperthermia: Potential and Limitations.

Hyperthermia, the mild elevation of temperature to 40-43°C, can induce cancer cell death and enhance the effects of radiotherapy and chemotherapy. However, achievement of its full potential as a clinically relevant treatment modality has been restricted by its inability to effectively and preferentially heat malignant cells. The limited spatial resolution may be circumvented by the intravenous administration of cancer-targeting magnetic nanoparticles that accumulate in the tumor, followed by the application of an alternating magnetic field to raise the temperature of the nanoparticles located in the tumor tissue. This targeted approach enables preferential heating of malignant cancer cells whilst sparing the surrounding normal tissue, potentially improving the effectiveness and safety of hyperthermia. Despite promising results in preclinical studies, there are numerous challenges that must be addressed before this technique can progress to the clinic. This review discusses these challenges and highlights the current understanding of targeted magnetic hyperthermia.

A photoelectrochemical platform for the capture and release of rare single cells.

For many normal and aberrant cell behaviours, it is important to understand the origin of cellular heterogeneity. Although powerful methods for studying cell heterogeneity have emerged, they are more suitable for common rather than rare cells. Exploring the heterogeneity of rare single cells is challenging because these rare cells must be first pre-concentrated and undergo analysis prior to classification and expansion. Here, a versatile capture & release platform consisting of an antibody-modified and electrochemically cleavable semiconducting silicon surface for release of individual cells of interest is presented. The captured cells can be interrogated microscopically and tested for drug responsiveness prior to release and recovery. The capture & release strategy was applied to identify rare tumour cells from whole blood, monitor the uptake of, and response to, doxorubicin and subsequently select cells for single-cell gene expression based on their response to the doxorubicin.

Development of New Gonadotropin-Releasing Hormone-Modified Dendrimer Platforms with Direct Antiproliferative and Gonadotropin Releasing Activity.

Gonadotropin-releasing hormone (GnRH) agonists (e.g., triptorelin) are used for androgen suppression therapy. They possess improved stability as compared to the natural GnRH, yet they suffer from a poor pharmacokinetic profile. To address this, we used a GnRH peptide-modified dendrimer platform with and without lipidation strategy. Dendrimers were synthesized on a polylysine core and bore either native GnRH (1, 2, and 5) or lipid-modified GnRH (3 and 4). Compound 3, which bore a lipidic moiety in a branched tetramer structure, showed approximately 10-fold higher permeability and metabolic stability and 39 times higher antitumor activity against hormone-resistant prostate cancer cells (DU145) relative to triptorelin. In gonadotropin-release experiments, dendrimer 3 was shown to be the most potent construct. Dendrimer 3 showed similar luteinizing hormone (LH)-release activity to triptorelin in mice. Our findings indicate that dendrimer 3 is a promising analog with higher potency for the treatment of hormone-resistant prostate cancer than the currently available GnRH agonists.

A study on liposomal encapsulation of a lipophilic prodrug of LHRH.

This study aimed at evaluating whether derivatization of luteinizing hormone-releasing hormone (LHRH) peptide with an amphiphilic lipoamino acid moiety could allow, along with other technological and/or pharmacokinetic advantages, to improve its encapsulation in liposomes, potentially driving its further body distribution and cellular uptake. Experimental data confirmed that a lipophilic derivative of LHRH was efficiently incorporated in various liposomal systems, differing in lipid composition and surface charge, and obtained using different methods of production. Incubation of liposomes, loaded with a fluorescent derivative of the LHRH prodrug, with NCTC keratinocytes or Caco-2 cell cultures showed that the carriers can be rapidly internalized. Conversely, the internalization of the free prodrug occurred only at very high concentrations.

Macromolecular Hydrogen Sulfide Donors Trigger Spatiotemporally Confined Changes in Cell Signaling.

Hydrogen sulfide (H2S) is involved in a myriad of cell signaling processes that trigger physiological events ranging from vasodilation to cell proliferation. Moreover, disturbances to H2S signaling have been associated with numerous pathologies. As such, the ability to release H2S in a cellular environment and stimulate signaling events is of considerable interest. Herein we report the synthesis of macromolecular H2S donors capable of stimulating cell signaling pathways in both the cytosol and at the cell membrane. Specifically, copolymers having pendent oligo(ethylene glycol) and benzonitrile groups were synthesized, and the benzonitrile groups were subsequently transformed into primary aryl thioamide groups via thionation using sodium hydrosulfide. These thioamide moieties could be incorporated into a hydrophilic copolymer or a block copolymer (i.e., into either the hydrophilic or hydrophobic domain). An electrochemical sensor was used to demonstrate release of H2S under simulated physiological conditions. Subsequent treatment of HEK293 cells with a macromolecular H2S donor elicited a slow and sustained increase in cytosolic ERK signaling, as monitored using a FRET-based biosensor. The macromolecular donor was also shown to induce a small, fast and sustained increase in plasma membrane-localized PKC activity immediately following addition to cells. Studies using an H2S-selective fluorescent probe in live cells confirmed release of H2S from the macromolecular donor over physiologically relevant time scales consistent with the signaling observations. Taken together, these results demonstrate that by using macromolecular H2S donors it is possible to trigger spatiotemporally confined cell signaling events. Moreover, the localized nature of the observed signaling suggests that macromolecular donor design may provide an approach for selectively stimulating certain cellular biochemical pathways.

Investigation of structure-activity relationships of synthetic anti-gonadotropin releasing hormone vaccine candidates.

The immunoneutralization of gonadotropin-releasing hormone (GnRH) can be used for the treatment of human hormone-dependent male and female cancers or as immunocontraceptives in animals. Vaccine candidates 1 [Th(K-LP)GnRH], 2 [GnRH(K-LP)Th], 3 [GnRH(K-Th)LP], and 4 [Th(K-GnRH)LP] (for which K=lysine, LP=lipopeptide Ser-Ser-C16 -C16 , and Th=T helper cell epitope influenza HA2), were synthesized by assembling a CD4(+) T helper cell epitope (Th), GnRH, and an adjuvanting lipid moiety (LP) in various spatial arrangements. All compounds were efficiently taken up by antigen-presenting cells with significant immunogenicity without an external adjuvant. Compounds 2, 3, and 4, in which GnRH is conjugated through its C terminus, produced higher GnRH-specific antibody responses than construct 1, in which the GnRH moiety is conjugated through its N terminus. All four constructs induced a significant antiproliferative effect (up to 55 %) on GnRH-receptor-positive LNCaP cells, but showed weaker activity in the GnRH-receptor-negative SKOV-3 cell line. Marked degenerative changes were observed in morphology and follicular development in the ovaries of immunized mice, with approximately 30 % higher degenerative antral and atretic follicles.

Active immunisation of mice with GnRH lipopeptide vaccine candidates: Importance of T helper or multi-dimer GnRH epitope.

Active immunisation against gonadotropin releasing hormone (GnRH) is a potential alternative to surgical castration. This study focused on the development of a GnRH subunit lipopeptide vaccine. A library of vaccine candidates that contained one or more (up to eight) copies of monomeric or dimeric GnRH peptide antigen, an adjuvanting lipidic moiety based on lipoamino acids, and an additional T helper epitope, was synthesised by solid phase peptide synthesis. The candidates were evaluated in vivo in order to determine the minimal components of this vaccine necessary to induce a systemic immune response. BALB/c mice were immunised with GnRH lipopeptide conjugates, co-administered with or without Complete Freund's Adjuvant, followed by two additional immunisations. Significant GnRH-specific IgG titres were detected in sera obtained from mice immunised with four of the seven lipopeptides tested, with an increase in titres observed after successive immunisations. This study highlights the importance of for epitope optimisation and delivery system design when producing anti-hapten antibodies in vivo. The results of this study also contribute to the development of future clinical and veterinary immunocontraceptives.

Calorimetry and Langmuir-Blodgett studies on the interaction of a lipophilic prodrug of LHRH with biomembrane models.

The interaction between an amphiphilic luteinizing hormone-releasing hormone (LHRH) prodrug that incorporated a lipoamino acid moiety (C12-LAA) with biological membrane models that consisted of multilamellar liposomes (MLVs) and phospholipid monolayers, was studied using Differential Scanning Calorimetry (DSC) and Langmuir-Blodgett film techniques. The effect of the prodrug C12[Q1]LHRH on the lipid layers was compared with the results obtained with the pure precursors, LHRH and C12-LAA. Conjugation of LHRH with a LAA promoiety showed to improve the peptide interaction with biomembrane models. Basing on the calorimetric findings, the LAA moiety aided the transfer of the prodrug from an aqueous solution to the biomembrane model.

Synthesis and in vitro evaluation of glycosyl derivatives of luteinizing hormone-releasing hormone (LHRH).

Luteinizing hormone-releasing hormone (LHRH) analogues are used extensively for the treatment of various hormone-dependent diseases. However, none of the currently marketed derivatives can be administered orally. Modification of peptide sequences by attachment of carbohydrate moieties is a promising strategy that may increase the metabolic stability of the target peptide and enhance its transport across cell membranes, subsequently improving peptide bioavailability. In this study, either the N- or C-terminus of the LHRH peptide was altered by attachment of carbohydrate moieties. Caco-2 cells were chosen as an in vitro model to investigate both the permeability and stability of the new LHRH analogues. Our findings show that conjugating sugar moieties to the N-terminus of the LHRH peptide significantly increased both permeability and metabolic stability of most of the modified LHRH derivatives.

Peripherally acting novel lipo-endomorphin-1 peptides in neuropathic pain without producing constipation.

We previously described two novel analogues of endomorphin-1 (Tyr-Pro-Trp-Phe-NH2, 1), modified with an 8-carbon lipoamino acid (C8LAA) with or without replacement of Tyr(1) with 2,6-dimethyltyrosine (Dmt) at the N-terminus of the peptide (compounds 3 and 4, respectively). They were shown to be more stable and permeable, and acted as potent µ-opioid receptor agonists. In this study we report that the C8LAA modification resulted in successful systemic delivery of both analogues. They produced potent dose-dependent pain relief in a chronic constriction injury-rat model of neuropathic pain after intravenous administration with ED50 values obtained at 6.58 (±1.22) µmol/kg for 3 and 6.18 (±1.17) µmol/kg for 4. Using two different rat models of constipation that assess the effects of µ-opioid receptor agonists on stool hydration and gastro-intestinal motility, compound 3 produced insignificant constipation at 16 µmol/kg, whereas morphine elicited significant constipation at 2 µmol/kg. Compound 3 in contrast to morphine, did not attenuate the hypercapnic ventilatory response at 5 µmol/kg, a dose that fully alleviated hindpaw sensitivity at the time of peak effect in CCI-rats. This finding revealed the lack of respiratory depression effect at antinociceptive dose.

Synthesis, biological activity and structure-activity relationship of endomorphin-1/substance P derivatives.

Endomorphins have been shown to produce potent analgesia in various rodent models of pain. However, their central administration led to the development of tolerance and physical dependence. Conjugation of C-terminal substance P (SP) fragments to opioids and opioid peptides was previously shown to produce hybrid peptides with strong analgesic activity, with low or no propensity to develop tolerance. In this study, four peptides (2-5) comprised of endomorphin-1 (1) and C-terminal fragments of SP (four or five amino acids, SP(8-11) (2) or SP(7-11) (4), respectively), with an overlapping Phe residue, were synthesized. To overcome low metabolic stability and poor membrane permeability of the peptide, the N-terminus of 2 and 4 was further modified with a C10-carbon lipoamino acid (C10LAA) achieving 3 and 5, respectively. LAA-modification of the hybrid peptides resulted in a significant increase in metabolic stability and membrane permeability compared to peptides 1, 2 and 4. Compound 5 showed potent µ-opioid receptor binding affinity (K(iµ)=3.87 ± 0.51 nM) with dose-dependent agonist activity in the nanomolar range (IC(50)=45 ± 13 nM). In silico modeling was used to investigate the binding modes and affinities of compounds 1-5 in the active site of µ-opioid receptors. The docking scores were in agreement with the K(iµ) values obtained in the receptor binding affinity studies. The more active LAA-modified hybrid peptide showed a lower total interaction energy and higher negative value of MolDock score.

Lipidated analogues of luteinizing hormone-releasing hormone (LHRH) reduce serum levels of follicle-stimulating hormone (FSH) after oral administration.

A diverse range of diseases involving the reproductive system are treated with luteinizing hormone-releasing hormone (LHRH) agonists which must be administered daily. Currently, an efficient oral delivery system is not available. Here, we show the facile inclusion of lipoamino acids into the peptide sequence of LHRH, rendering it more stable towards enzymatic degradation, as well as enhancing permeability across Caco-2 cell monolayers. Selected LHRH derivatives were tested in vivo by daily oral administration to rats. The size and weight of the sex organs remained unchanged and the levels of LH were stable over the course of the experiment. However, some of the lipidic peptides (3, 8 and 9) were able to reduce serum levels of follicle-stimulating hormone (FSH), an important finding towards the development of orally available LHRH agonists.