Doxorubicin Conjugates Research Articles

The Product of Matrix Metalloproteinase Cleavage of Doxorubicin Conjugate for Anticancer Drug Delivery: Calorimetric, Spectroscopic, and Molecular Dynamics Studies on Peptide-Doxorubicin Binding to DNA.

Matrix metalloproteinases (MMPs) are extracellular matrix degradation factors, promoting cancer progression. Hence, they could provide an enzyme-assisted delivery of doxorubicin (DOX) in cancer treatment. In the current study, the intercalation process of DOX and tetrapeptide–DOX, the product of the MMPs’ cleavage of carrier-linked DOX, into dsDNA was investigated using stationary and time-resolved fluorescence spectroscopy, UV-Vis spectrophotometry and isothermal titration calorimetry (ITC). The molecular dynamics (MD) simulations on the same tetrapeptide–DOX…DNA and DOX…DNA systems were also performed. The undertaken studies indicate that DOX and tetrapeptide–DOX can effectively bond with dsDNA through the intercalation mode; however, tetrapeptide–DOX forms less stable complexes than free DOX. Moreover, the obtained results demonstrate that the differences in DNA affinity of both forms of DOX can be attributed to different intercalation modes. Tetrapeptide–DOX shows a preference to intercalate into DNA through the major groove, whereas DOX does it through the minor one. In summary, we can conclude that the tetrapeptide–DOX intercalation to DNA is significant and that even the lack of non-specific proteases releasing DOX from the tetrapeptide conjugate, the presence of which is suggested by the literature for the efficient release of DOX, should not prevent the cytostatic action of the anthracycline.

Doxorubicin Loaded Nanoparticle Consisting of Chitosan and Mannose Modified Graphene Oxide for Intracellular Drug Delivery and Anti-tumor Activity

In order to develop the efficiency and the specificity of anticancer drug delivery, we have designed an innovative nanocarrier. The nanocarrier system comprises of a multifunctional graphene oxide nanoparticle-based drug delivery system (GO-CS-M-DOX) as a novel platform for intracellular drug delivery of doxorubicin (DOX). Firstly, graphene oxide (GO) was synthesized by hummer’s method whose surface was functionalized by chitosan (CS) in order to obtain a more precise drug delivery, the system was then decorated with mannose (M). Further conjugation of an anti-cancer drug doxorubicin to the nanocarrier system resulted in GO-CS-M-DOX drug delivery system. The resultant conjugate was characterized for its physio-chemical properties and its biocompatibility was evaluated via hemolysis assay. The drug entrapment efficiency is as high as 90% and in vitro release studies of DOX under pH 5.3 is significantly higher than that under pH 7.4. The anticancer activity of the synthesized drug delivery system was studied by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay against MCF-7 cell line. These results stated that the pH dependent multifunctional doxorubicin- chitosan functionalized graphene oxide based nanocarrier system, could lead to a promising and potential platform for intracellular delivery and cytotoxicity activity for variety of anticancer drugs. 

Doxorubicin Conjugation to Reovirus Improves Oncolytic Efficacy in Triple-Negative Breast Cancer

Breast cancer is the second leading cause of cancer-related deaths in women in the United States. The triple-negative breast cancer (TNBC) subtype associates with higher rates of relapse, shorter overall survival, and aggressive metastatic disease. Hormone therapy is ineffective against TNBC, leaving patients with limited therapeutic options. Mammalian orthoreovirus (reovirus) preferentially infects and kills transformed cells, and a genetically engineered reassortant reovirus infects and kills TNBC cells more efficiently than prototypical strains. Reovirus oncolytic efficacy is further augmented by combination with topoisomerase inhibitors, including the frontline chemotherapeutic doxorubicin. However, long-term doxorubicin use correlates with toxicity to healthy tissues. Here, we conjugated doxorubicin to reovirus (reo-dox) to control drug delivery and enhance reovirus-mediated oncolysis. Our data indicate that conjugation does not impair viral biology and enhances reovirus oncolytic capacity in TNBC cells. Reo-dox infection promotes innate immune activation, and crosslinked doxorubicin retains DNA-damaging properties within infected cells. Importantly, reovirus and reo-dox significantly reduce primary TNBC tumor burden in vivo, with greater reduction in metastatic burden after reo-dox inoculation. Together, these data demonstrate that crosslinking chemotherapeutic agents to oncolytic viruses facilitates functional drug delivery to cells targeted by the virus, making it a viable approach for combination therapy against TNBC.

Functionalized Block Co‐Polymer Pro‐Drug Nanoparticles with Anti‐Cancer Efficacy in 3D Spheroids and in an Orthotopic Triple Negative Breast Cancer Model

AbstractAmphiphilic block co‐polymers composed of poly(ethylene glycol)‐co‐poly(lactide)‐co‐poly(2‐((tert‐butoxycarbonyl)amino)‐3‐propyl carbonate) (PEG‐pLA‐pTBPC) are synthesized in monomer ratios and arrangements to enable assembly into nanoparticles with different sizes and architectures. These materials are based on components in clinical use, or known to be biodegradable, and retain the same fundamental chemistry across “AB” and “BAB” block architectures. In MCF7 and MDA‐MB‐231 breast cancer cells, nanoparticles of <100 nm are internalized most rapidly, by both clathrin‐ and caveolin‐mediated pathways. In THP‐1 cells, polymer architecture and length of the hydrophilic block is the most important factor in the rate of internalization. The organ distributions of systemically injected nanoparticles in healthy mice indicate highest accumulation of the BAB‐blocks in lungs and liver and the lowest accumulation in these organs of a methoxyPEG5000‐pLA‐pTBPC polymer. Conjugation of doxorubicin via a serum‐stable urea linker to the carbonate regions of PEG5000‐pLA‐pTBPC generates self‐assembling nanoparticles which are more cytotoxic in 2D, and penetrate further in 3D spheroids of triple negative breast cancer cells, than the free drug. In an aggressive orthotopic triple negative breast cancer mouse model, the methoxyPEG5000‐pLA‐pTBPC is of similar potency to free doxorubicin but with no evidence of adverse effects in terms of body weight.

Doxorubicin-Conjugated Innovative 16-mer DNA Aptamer-Based Annexin A1 Targeted Anti-Cancer Drug Delivery

Aptamers are small, functional single-stranded DNA or RNA oligonucleotides that bind to their targets with high affinity and specificity. Experimentally, aptamers are selected by the systematic evolution of ligands by exponential enrichment (SELEX) method. Here, we have used rational drug designing and bioinformatics methods to design the aptamers, which involves three different steps. First, finding a probable aptamer-binding site, and second, designing the recognition and structural parts of the aptamers by generating a virtual library of sequences, selection of specific sequence via molecular docking, molecular dynamics (MD) simulation, binding energy calculations, and finally evaluating the experimental affinity. Following this strategy, a 16-mer DNA aptamer was designed for Annexin A1 (ANXA1). In a direct binding assay, DNA1 aptamer bound to the ANXA1 with dissociation constants value of 83 nM. Flow cytometry and fluorescence microscopy results also showed that DNA1 aptamer binds specifically to A549, HepG2, U-87 MG cancer cells that overexpress ANXA1 protein, but not to MCF7 and L-02, which are ANXA1 negative cells. We further developed a novel system by conjugating DNA1 aptamer with doxorubicin and its efficacy was studied by cellular uptake and cell viability assay. Also, anti-tumor analysis showed that conjugation of doxorubicin with aptamer significantly enhances targeted therapy against tumors while minimizing overall adverse effects on mice health.

Fabrication of unimolecular micelle-based nanomedicines from hyperbranched polymers containing both terminal and internal reactive groups

Unimolecular micelles are an attractive type of nanocarriers for drug delivery, but developing unimolecular micelle-based nanomedicine with high drug loading capacity and excellent unimolecular stability is challenging. Herein, we report the design and synthesis of hyperbranched polymers containing both terminal and internal reactive groups for fabricating the unimolecular micelle-based nanocarriers with high drug loading ability and superior unimolecular stability using selective modification. Specifically, we synthesized hyperbranched poly(amino ester)s containing terminal acrylate groups and internal R groups (named HBPAE-R) via the Michael addition polymerization of A2 + BʹB2-R monomers, wherein A represents acrylate, and B, Bʹ represents the hydrogen in primary amine and secondary amine group, respectively, whereas R represents hydroxy, alkene or alkyne groups that do not participate in the polymerization process but assist in additional modification. Then, poly(ethylene glycol) (PEG) chains were linked to the surface of HBPAE-OH (representative molecules) with the subsequent conjugation of doxorubicin (DOX, representative drug) to the internal hydroxy groups through an acid-labile hydrazide bond, to afford anti-cancer drug-loaded hyperbranched star copolymers PEG-HBPAE-DOX. The obtained nanomedicines exhibited good unimolecular stability, relatively high drug loading content and a controlled drug release behavior. The in vitro cytotoxicity experiments further demonstrated the anti-tumor efficacy of these unimolecular nanomedicines. Our approach is expected to further boost research on the fabrication of multifunctional unimolecular nanomedicines from hyperbranched polymers containing both terminal and internal reactive groups.

Conjugation of Doxorubicin to siRNA Through Disulfide-based Self-immolative Linkers.

Co-delivery systems of siRNA and chemotherapeutic drugs have been developed as an attractive strategy to optimize the efficacy of chemotherapy towards cancer cells with multidrug resistance. In these typical systems, siRNAs are usually associated to drugs within a carrier but without covalent interactions with the risk of a premature release and degradation of the drugs inside the cells. To address this issue, we propose a covalent approach to co-deliver a siRNA-drug conjugate with a redox-responsive self-immolative linker prone to intracellular glutathione-mediated disulfide cleavage. Herein, we report the use of two disulfide bonds connected by a pentane spacer or a p-xylene spacer as self-immolative linker between the primary amine of the anticancer drug doxorubicin (Dox) and the 2′-position of one or two ribonucleotides in RNA. Five Dox-RNA conjugates were successfully synthesized using two successive thiol-disulfide exchange reactions. The Dox-RNA conjugates were annealed with their complementary strands and the duplexes were shown to form an A-helix sufficiently stable under physiological conditions. The enzymatic stability of Dox-siRNAs in human serum was enhanced compared to the unmodified siRNA, especially when two Dox are attached to siRNA. The release of native Dox and RNA from the bioconjugate was demonstrated under reducing conditions suggesting efficient linker disintegration. These results demonstrate the feasibility of making siRNA-drug conjugates via disulfide-based self-immolative linkers for potential therapeutic applications.

Synthesis and evaluation of new peptide-linked doxorubicin conjugates as prodrugs activated by prostate-specific antigen

The targeted delivery of cytotoxic agents to prostate cancer cells via selective activation of peptide-linked prodrugs by prostate-specific antigen (PSA) has been previously demonstrated. In our continued efforts to design prodrugs with improved prostate tumor specificity, we developed GABA ← mGly-Ala-Ser-Chg-Gln and glutaryl-Ser-Ala-Ser-Chg-Gln as promoieties with enhanced PSA specificity starting from the known substrate sequence glutaryl-Hyp-Ala-Ser-Chg-Gln. Based on their PSA cleavage rates and resistance to non-PSA-mediated hydrolysis in plasma, GABA ← mGly-Ala-Ser-Chg-Gln and glutaryl-Ser-Ala-Ser-Chg-Gln were selected as optimal promoieties and coupled to doxorubicin (Dox) as PSA-targeted prodrugs, using Ser-Leu linkers. Following 72-h incubations with Dox prodrugs, there was insignificant cytotoxicity in non-PSA-producing DU145 cells. The Dox prodrugs, glutaryl-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-Dox (I), glutaryl-Ser-Ala-Ser-Chg-Gln-Ser-Leu-Dox (II) and GABA ← mGly-Ala-Ser-Chg-Gln-Ser-Leu-Dox (III) demonstrated comparable PSA cleavage rates (t1/2 values <23 min), and were cytotoxic against PSA-producing LNCaP cells with IC50 values of 0.18, 0.27 and 0.082 μM, respectively. To mitigate neprilysin-mediated hydrolysis of the Ser-Leu linker in prodrugs I–III and further improve PSA specificity, 3-aminooxypropionate was incorporated between the promoiety and Dox (prodrug IV). Despite its slower PSA cleavage rate (t1/2 value of 67 min), GABA ← mGly-Ala-Ser-Chg-Gln-NH-O-CH2-C(Me)2C(O)-14-O-Dox (IV) was equipotent (IC50 value of 0.19 μM) to prodrug I at killing PSA-producing LNCaP cells due to its ability to release free Dox through a cyclization activation mechanism. Further metabolism and PK/PD studies will be conducted to evaluate the tumor specificity of the novel Dox prodrugs (II–IV) reported herein.

In vitro vascular toxicity assessment of NitDOX, a novel NO-releasing doxorubicin

The conjugation of doxorubicin (DOX) with nitric oxide (NO)-releasing groups gave rise to novel anthracyclines, such as nitrooxy-DOX (NitDOX), capable to overcome multidrug resistance. The widely described anthracycline cardiovascular toxicity, however, might limit their clinical use. This study aimed to investigate NitDOX-induced effects, as potential hazard, on vascular smooth muscle A7r5 and endothelial EA.hy926 cell viability, on the mechanical activity of freshly and cultured rat aorta rings, as well as on Cav1.2 channels of A7r5 cells. DOX was used as a reference compound.Although an increase in intracellular radicals and a reduction in mitochondrial potential occurred upon treatment with both drugs, A7r5 and EA.hy926 cells proved to be more sensitive to DOX than to NitDOX. Both compounds promoted comparable effects in A7r5 cells, whereas NitDOX was less active than DOX in inducing DNA damage and in eliciting apoptotic-mediated cell death revealed as an increase in sub-diploid-, DAPI- and annexin V-positive- EA.hy926 cell percentage. Moreover, in EA.hy926 cells, NitDOX doubled basal NO content, while preincubation with the NO-scavenger PTIO increased NitDOX-induced cytotoxicity.DOX exhibited a negligible contracturing effect in endothelium-intact rings, while NitDOX induced a significant ODQ-sensible, vasodilation in endothelium-denuded rings. In arteries cultured with both drugs for 7 days, NitDOX prevented either phenylephrine- or KCl-induced contraction at a concentration 10-fold higher than that of DOX. These results demonstrate that NitDOX displays a more favourable vascular toxicity profile than DOX. Taking into account its greater efficacy against drug-resistant cells, NitDOX is worth of further investigations in preclinical and clinical settings.

Switchable Fluorescence of Doxorubicin for Label-Free Imaging of Bioorthogonal Drug Release.

Monitoring the release and activation of prodrug formulations provides essential information about the outcome of a therapy. While the prodrug delivery can be confirmed by using different imaging techniques, confirming the release of active payload by using imaging is a challenge. Here, we have discovered that the switchable fluorescence of doxorubicin can validate drug release upon its uncaging reaction with a highly specific chemical partner. We have observed that the conjugation of doxorubicin with a trans-cyclooctene (TCO) diminishes its fluorescence at 595 nm. This quenched fluorescence of the doxorubicin prodrug is recovered upon its bond-cleaving reaction with tetrazine. Clinically assessed iron oxide nanoparticles were used to formulate a doxorubicin nanodrug. The release of doxorubicin from the nanodrug was studied under various experimental conditions. A fivefold increase in doxorubicin fluorescence is observed after complete release. The studies were carried out in vitro in MDA-MB-231 breast cancer cells. An increase in Dox signal was observed upon tetrazine administration. This switchable fluorescence mechanism of Dox could be employed for fundamental studies, that is, the reactivity of various tetrazine and TCO linker types under different experimental conditions. In addition, the system could be instrumental for translational research where the release and activation of doxorubicin prodrug payloads can be monitored by using optical imaging systems.

Bifunctional Aptamer-Doxorubicin Conjugate Crosses the Blood-Brain Barrier and Selectively Delivers Its Payload to EpCAM-Positive Tumor Cells.

The prognosis for breast cancer patients diagnosed with brain metastases is poor, with survival time measured merely in months. This can largely be attributed to the limited treatment options capable of reaching the tumor as a result of the highly restrictive blood–brain barrier (BBB). While methods of overcoming this barrier have been developed and employed with current treatment options, the majority are highly invasive and nonspecific, leading to severe neurotoxic side effects. A novel approach to address these issues is the development of therapeutics targeting receptor-mediated transport mechanisms on the BBB endothelial cell membranes. Using this approach, we intercalated doxorubicin (DOX) into a bifunctional aptamer targeting the transferrin receptor on the BBB and epithelial cell adhesion molecule (EpCAM) on metastatic cancer cells. The ability of the DOX-loaded aptamer to transcytose the BBB and selectively deliver the payload to EpCAM-positive tumors was evaluated in an in vitro model and confirmed for the first time in vivo using the MDA-MB-231 breast cancer metastasis model (MDA-MB-231Br). We show that colocalized aptamer and DOX are clearly detectable within the brain lesions 75 min postadministration. Collectively, results from this study demonstrate that through intercalation of a cytotoxic drug into the bifunctional aptamer, a therapeutic delivery vehicle can be developed for specific targeting of EpCAM-positive brain metastases.

Zwitterionic Polymer Micelles with Dual Conjugation of Doxorubicin and Curcumin: Synergistically Enhanced Efficacy against Multidrug-Resistant Tumor Cells.

This paper reports a novel redox-sensitive micellar system for the co-delivery of doxorubicin (Dox) and a chemosensitizer (curcumin, Cur) to overcome the multidrug resistance (MDR) in cancer cells. Dox and Cur were co-conjugated onto a zwitterionic polymer, poly(carboxybetaine) (pCB), to form Cur-pCB-Dox that self-assembled into stable micelles (164.2 ± 4.8 nm). Single-drug conjugates (pCB-Dox and pCB-Cur) were prepared for comparisons. Compared to the high half-maximal inhibitory concentration (IC50) of Dox (437.2 μg/mL), the IC50 value of pCB-Dox (14.1 μg/mL) was only 1/33 that of Dox. Confocal laser scanning microscopy and flow cytometry revealed the greatly enhanced cell uptake of the conjugate due to the enhanced permeability and retention effect of tumor cells on the micellar conjugate. Co-delivery of pCB-Dox with pCB-Cur further reduced the IC50 value by 37% (8.9 μg/mL). More importantly, Cur-pCB-Dox exhibited the strongest cytotoxicity against MCF-7/Adr cells (IC50, 5.87 μg/mL) because the co-delivered Dox and Cur on one carrier specifically transported into the same cells, which inhibited the efflux of Dox by Cur, led to a higher intracellular Dox concentration and made the drugs exert synergistic effects at the targeting regions. The results proved the zwitterionic micelles as promising drug co-delivery vehicles for fighting against MDR.

Tumor-specific fluorescent Cdots-based nanotheranostics by acid-labile conjugation of doxorubicin onto reduction-cleavable Cdots-based nanoclusters.

Carbon quantum dots (Cdots) have attracted more and more interests in bioimaging and tumor theranostics. However, their practical application has been limited due to the small particle size and non-tumor-specific fluorescence. Here, reduction-cleavable disulfide-linked Cdots-based nanoclusters were fabricated to conjugate doxorubicin (DOX) via an acid-labile hydrazone bond. Owing to the pH and reduction dual-stimuli responsiveness, the proposed Cdots-based nanotheranostics possessed unique tumor-specific fluorescent property and tumor-specific controlled drug release performance, indicating their promising potential for the in-situ real-time fluorescent monitoring of therapeutic response in future tumor therapy.

Multi pH-sensitive polymer-drug conjugate mixed micelles for efficient co-delivery of doxorubicin and curcumin to synergistically suppress tumor metastasis.

Combination therapy has been proved to be an effective strategy to inhibit metastasis, however, its efficacy is always compromised by the poor delivery efficiency of drugs. In this study, multi pH-sensitive polymer-drug conjugate mixed micelles were fabricated by the self-assembly of PEOz-PLA-ace-Cur, a conjugate of curcumin (Cur) with poly(2-ethyl-2-oxazoline)-poly(d,l-lactide) (PEOz-PLA) through the linkage of the pH-cleavable acetal bond, and PEOz-PLA-imi-DOX, a conjugate of doxorubicin (DOX) with PEOz-PLA through the linkage of the pH-cleavable benzoic imine bond. The mixed conjugate micelles (PP-Cur/PP-DOX-Mix-PMs) with accurately and conveniently controlled mass ratio of the two drugs were demonstrated to have a small particle size (40-128 nm), high drug loading capacity and pH-dependent drug release behavior. Notably, PP-Cur5/PP-DOX1-Mix-PMs exhibited slower DOX release under physiological conditions compared with PEOz-PLA-imi-DOX micelles, resulting in deeply reduced side effects in vivo. Furthermore, the mixed conjugate micelles showed synergistically enhanced inhibition of MDA-MB-231 cell growth and metastasis evidenced by the results of in vitro anti-invasion, wound healing and anti-migration assessment, and in vivo bioluminescence imaging in nude mice, and significant reduction of the side effects of DOX compared with dual drug physically loaded polymeric micelles. Mechanistic studies demonstrated that the possible inhibitory mechanism of PP-Cur5/PP-DOX1-Mix-PMs on tumor metastasis could be assigned to their inhibition of the invasion, migration, intravasation and extravasation of tumor cells. In conclusion, the multi pH-sensitive polymer-drug conjugate mixed micelles with synergistically enhanced anti-tumor and anti-metastasis activity are potential candidates for safe and effective cancer combination therapy.

Morphology tunable and acid-sensitive dextran-doxorubicin conjugate assemblies for targeted cancer therapy.

Stimuli-responsive and targetable nanomedicine systems have been widely applied as effective modalities for drug delivery and tumor therapeutics. Particle shape is also important for the biodistribution and cellular uptake in drug delivery applications. Here, morphology tunable and acid-responsive dextran-doxorubicin conjugate assemblies of DD-M and DDF-V for targeted doxorubicin (DOX) delivery were constructed, which contain the following favorable advantages: (1) one-pot synthesis of the drug loaded system with a Schiff base reaction is a green chemistry method which is better than the conventional drug conjugation/encapsulation methods. (2) The morphology of the nanoparticles could be regulated from a micelle (DD-M) to vesicle (DDF-V) structure by either introducing folic acid (FA) or not. (3) The abundant hydroxyl groups and electronegativity give DD-M and DDF-V superior stability in the physiological environment. (4) Besides, the multifunctional DDF-V with its important merits including tumor-targeting ability and acid-responsiveness is specific for DOX delivery in cancer therapy. (5) Compared to free DOX and DD-M, DDF-V displayed enhanced anti-tumor efficacy both in vitro and in vivo without obvious systematic toxicity. The morphology tunable, acid-sensitive and targetable nanosystem could be a promising strategy for site-specific drug delivery and potential cancer therapy in the future.

Poly[N-(2-hydroxypropyl)methacrylamide]-Modified Magnetic γ-F2 O3 Nanoparticles Conjugated with Doxorubicin for Glioblastoma Treatment.

With the aim to develop a new anticancer agent, we prepared poly[N-(2-hydroxypropyl)methacrylamide-co-methyl 2-methacrylamidoacetate] [P(HP-MMAA)], which was reacted with hydrazine to poly[N-(2-hydroxypropyl)methacrylamide-co-N-(2-hydrazinyl-2-oxoethyl)methacrylamide] [P(HP-MAH)] to conjugate doxorubicin (Dox) via hydrazone bond. The resulting P(HP-MAH)-Dox conjugate was used as a coating of magnetic γ-Fe2 O3 nanoparticles obtained by the coprecipitation method. In vitro toxicity of various concentrations of Dox, P(HP-MAH)-Dox, and γ-Fe2 O3 @P(HP-MAH)-Dox nanoparticles was determined on somatic healthy cells (human bone marrow stromal cells hMSC), human glioblastoma line (GaMG), and primary human glioblastoma (GBM) cells isolated from GBM patients both at a short and prolonged exposition time (up to 7 days). Due to hydrolysis of the hydrazone bond in acid milieu of tumor cells and Dox release, the γ-Fe2 O3 @P(HP-MAH)-Dox nanoparticles significantly decreased the GaMG and GBM cell growth compared to free Dox and P(HP-MAH)-Dox in low concentration (10 nM), whereas in hMSCs it remained without effect. γ-F2 O3 @PHP nanoparticles alone did not affect the viability of any of the tested cells.

EXTH-69. NANOTHERAPEUTICS FOR NEUROSURGICALLY-APPLIED DRUG DELIVERY

Abstract Despite multimodal treatment, the median survival of Glioblastoma multiforme (GBM) patients remains less than 15 months, in considerable part due to diffusely infiltrative disease. Better treatment methods are necessary to eradicate residual tumour burden remaining beyond the resection cavity boundary. It is hypothesised that incorporating drug-loaded polymer pro-drug nanoparticles into a biodegradable microparticulate paste will lead to efficacious local delivery. We report the formulation of numerous self-assembling cytocompatible nanoparticles, based on different linear and branched polymeric architectures, amenable for localised intra-cavity delivery post-surgery. The polymers were synthesised by ring-opening polymerisation with organic catalysts, leading to controlled reaction kinetics and greater potential biomedical applicability. A simple nanoprecipitation technique was employed to gain nanoparticles with the size range of 60–130 nm, depending on the initial monomer ratios and polymeric architectures. Successful biocompatibility studies of the self-assembling nanoparticles have been carried out in vitro on the U87 glioma cell line. We demonstrated that copolymerisation of a monomer with functional capability enabled the successful conjugation of doxorubicin to the polymer chain. We will discuss strategies to incorporate pH-sensitive linkers to the polymeric backbone, which would allow controlled drug release in acidic microenvironments. Based on an increasing understanding of GBM intra-tumour heterogeneity, the capability to deliver multiple therapeutic moieties from single formulations is clinically-relevant. Thus, we hypothesised that polymers with a greater degree of branching over traditional linear structures would lead to greater drug loading, and successfully tested this hypothesis through the encapsulation of olaparib. Future work will assess the efficacy of the polymer pro-drug nanoparticles against both commercial and primary cell lines, and safety/efficacy of intra-cavity delivery using orthotopic syngeneic allografts, thus giving a more therapeutically-relevant insight into the activity of the formulations. This is the first study incorporating polymer pro-drugs of this type into an existing localised micro-scale delivery system for GBM therapies.

Intracellular trafficking and cytotoxicity of a gelatine–doxorubicin conjugate in two breast cancer cell lines

Details of intracellular pathways of cytotoxicity remain unclear for doxorubicin conjugates being studied to treat breast cancer tumours. A high molecular weight gelatine–doxorubicin conjugate was investigated with an emphasis on lysosome participation. The conjugate was synthesised and characterised. Cell uptake and cellular localisation in MCF-7 and triple negative breast cancer (TNBC) MDA-MB-231 cells were determined with fluorescence microscopy. Nuclear content of released DOX was determined by UHPLC. Cytotoxicity was determined by the MTT assay. Lysosome membrane permeabilization (LMP) was followed by lysosomal release of fluorescently labelled dextran. After incubation at an equivalent 10 µM DOX, conjugate lysosome accumulation was substantial in both cell lines by 24 h, at which time the conjugate cytotoxic effect was first observed. By 48 h, the conjugate was nearly fourfold more toxic in TNBC than in MCF-7 cells. The MCF-7 nucleus drug content from conjugate released DOX was small but confirmed intra-lysosomal drug release. The conjugate induced LMP in 100% of TNBC cells but LMP was virtually absent in MCF-7 cells. These results suggest that the conjugate induces cytotoxicity by a lysosomal pathway in MDA-MB-231 cells and has potential for treatment of TNBC tumours. Support: NIH/NCI R15CA135421, the Agnes Varis Trust for Women’s Health.

Doxorubicin and Varlitinib Delivery by Functionalized Gold Nanoparticles Against Human Pancreatic Adenocarcinoma.

The aim of this study was to develop drug delivery nanosystems based on pegylated gold nanoparticles (PEGAuNPs) for a combination against pancreatic cancer cells. Doxorubicin and varlitinib, an anthracycline and a tyrosine kinase inhibitor respectively, were conjugated with gold nanoparticles. The systems were characterized, after synthesis, regarding their size, stability and morphology. An efficient conjugation of doxorubicin and varlitinib with PEGAuNPs was revealed. The cytotoxicity effect induced by the combination of the nanoconjugates was investigated in pancreatic cancer cell lines. Doxorubicin and varlitinib conjugated with PEGAuNPs revealed a combined effect to decrease the cell survival of the cancer line S2-013s, while reducing the drugs’ toxicity for the healthy pancreatic cells hTERT-HPNE. This study highlights the promising potential of PEGAuNPs for targeted delivery of therapeutic drugs into human cells, enhancing the antitumor growth-inhibition effect on cancer cells, and decreasing the toxicity against normal cells. In cancer therapy, the present approach based on PEGAuNP functionalization can be further explored to increase drug targeting efficiency and to reduce side effects.

Nanotherapeutics for neurosurgically-applied drug delivery

Abstract Despite multimodal treatment, the median survival of Glioblastoma multiforme (GBM) remains less than 15 months, in considerable part due to diffusely infiltrative disease. Better treatment methods are necessary to eradicate residual tumour burden remaining beyond the resection cavity boundary. Based on an increasing understanding of GBM intra-tumour heterogeneity, the capability to deliver multiple therapeutic moieties from single formulations is clinically-relevant. It is hypothesised that incorporating drug-loaded polymer pro-drugs, which are capable of transcytotic ‘hopping’, into a biodegradable microparticulate paste will lead to efficacious local delivery. Here we report the formulation of numerous self-assembling cytocompatible nanoparticles, based on different linear and branched polymeric architectures. The polymers were synthesised by ring opening polymerisation with organic catalysts, leading to controlled reaction kinetics and greater potential biomedical applicability. We demonstrated that copolymerisation of a monomer with functional capability enabled the successful conjugation of doxorubicin to the polymer chain. We hypothesised that polymers with a greater degree of branching over traditional linear structures would lead to greater drug loading, and successfully tested this hypothesis through the encapsulation of olaparib. We will discuss strategies to incorporate: i) pH-sensitive linkers to the polymeric backbone, which would allow controlled drug release in acidic microenvironments; ii) multiple combined chemotherapeutics, including doxorubicin and olaparib. Future work will assess the efficacy of the polymer pro-drugs against primary GBM lines derived from the invasive margin and safety/efficacy using orthotopic syngeneic allografts. This is the first study incorporating polymer pro-drugs of this type into an existing localised micro-scale delivery system for GBM therapies.