ABSTRACT
The clinical prospects of cancer nanomedicines depend on effective patient stratification. Here we report the identification of predictive biomarkers of the accumulation of nanomedicines in tumour tissue. By using supervised machine learning on data of the accumulation of nanomedicines in tumour models in mice, we identified the densities of blood vessels and of tumour-associated macrophages as key predictive features. On the basis of these two features, we derived a biomarker score correlating with the concentration of liposomal doxorubicin in tumours and validated it in three syngeneic tumour models in immunocompetent mice and in four cell-line-derived and six patient-derived tumour xenografts in mice. The score effectively discriminated tumours according to the accumulation of nanomedicines (high versus low), with an area under the receiver operating characteristic curve of 0.91. Histopathological assessment of 30 tumour specimens from patients and of 28 corresponding primary tumour biopsies confirmed the score's effectiveness in predicting the tumour accumulation of liposomal doxorubicin. Biomarkers of the tumour accumulation of nanomedicines may aid the stratification of patients in clinical trials of cancer nanomedicines.
ABSTRACT
Combinations of the topoisomerase II inhibitor doxorubicin and the poly (ADP-ribose) polymerase inhibitor olaparib offer potential drug-drug synergy for the treatment of triple negative breast cancers (TNBC). In this study we performed in vitro screening of combinations of these drugs, administered directly or encapsulated within polymer nanoparticles, in both 2D and in 3D spheroid models of breast cancer. A variety of assays were used to evaluate drug potency, and calculations of combination index (CI) values indicated that synergistic effects of drug combinations occurred in a molar-ratio dependent manner. It is suggested that the mechanisms of synergy were related to enhancement of DNA damage as shown by the level of double-strand DNA breaks, and mechanisms of antagonism associated with mitochondrial mediated cell survival, as indicated by reactive oxygen species (ROS) generation. Enhanced drug delivery and potency was observed with nanoparticle formulations, with a greater extent of doxorubicin localised to cell nuclei as evidenced by microscopy, and higher cytotoxicity at the same time points compared to free drugs. Together, the work presented identifies specific combinations of doxorubicin and olaparib which were most effective in a panel of TNBC cell lines, explores the mechanisms by which these combined agents might act, and shows that formulation of these drug combinations into polymeric nanoparticles at specific ratios conserves synergistic action and enhanced potency in vitro compared to the free drugs.
Subject(s)
Antineoplastic Agents , Nanoparticles , Phthalazines , Piperazines , Triple Negative Breast Neoplasms , Humans , Triple Negative Breast Neoplasms/metabolism , Reactive Oxygen Species , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Doxorubicin/pharmacology , Doxorubicin/therapeutic use , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Drug Combinations , Cell Line, TumorABSTRACT
Here, we aimed to chemically modify PAMAM dendrimers using lysine as a site-selective anchor for successfully delivering mRNA while maintaining a low toxicity profile. PAMAM dendrimers were multi-functionalised by amidation reactions in a regioselective, quantitative and stepwise manner with carefully selected property-modifying surface groups. Alternatively, novel lysine-based dendrimers were prepared in the same manner with the aim to unlock their potential in gene delivery. The modified dendrimers were then formulated with Cy5-EGFP mRNA by bulk mixing via liquid handling robotics across different nitrogen to phosphate ratios. The resulting dendriplexes were characterised by size, charge, mRNA encapsulation, and mRNA binding affinity. Finally, their in-vitro delivery activity was systematically investigated across key cellular trafficking stages to relate chemical design to cellular effect. We demonstrate our findings in different cell lines and benchmarked relative to a commercially available transfection agent, jetPEI®. We demonstrate that specific surface modifications are required to generate small, reliable and well-encapsulated positively charged dendriplex complexes. Furthermore, we show that introduction of fusogenic groups is essential for driving endosomal escape and achieving cellular delivery and translation of mRNA in these cell lines.
Subject(s)
Dendrimers , Dendrimers/chemistry , Polylysine , Transfection , Gene Transfer TechniquesABSTRACT
The recent emergence of drug-dendrimer conjugates within pharmaceutical industry research and development introduces a range of challenges for analytical and measurement science. These molecules are very high molecular weight (100-200kDa) with a significant degree of structural complexity. The characteristics and quality attributes that require understanding and definition, and impact efficacy and safety, are diverse. They relate to the intact conjugate, the various building blocks of these complex systems and the level of the free and bound active pharmaceutical ingredient (API). From an analytical and measurement science perspective, this necessitates the measurement of the molecular weight, impurity characterisation, the quantitation of the number of conjugated versus free API molecules, the determination of the impurity profiles of the building blocks, primary structure and both particle size and morphology. Here we report the first example of a global characterisation of a drug-dendrimer conjugate - PEGylated poly-lysine dendrimer currently under development (AZD0466). The impact of the wide variety of analytical and measurement techniques on the overall understanding of this complex molecular entity is discussed, with the relative capabilities of the various approaches compared. The results of this study are an essential platform for the research and development of the future generations of related dendrimer-based medicines.
Subject(s)
Antineoplastic Agents , Dendrimers , Dendrimers/chemistry , Lysine , Antineoplastic Agents/chemistry , Polyethylene Glycols/chemistryABSTRACT
As a malignant tumour of lymphatic origin, B-cell lymphoma represents a significant challenge for drug delivery, where effective therapies must access malignant cells in the blood, organs and lymphatics while avoiding off-target toxicity. Subcutaneous (SC) administration of nanomedicines allows preferential access to both the lymphatic and blood systems and may therefore provide a route to enhanced drug exposure to lymphomas. Here we examine the impact of SC dosing on lymphatic exposure, pharmacokinetics (PK), and efficacy of AZD0466, a small molecule dual Bcl-2/Bcl-xL inhibitor conjugated to a 'DEP®' G5 poly-l-lysine dendrimer. PK studies reveal that the plasma half-life of the dendrimer-drug conjugate is 8-times longer than that of drug alone, providing evidence of slow release from the circulating dendrimer nanocarrier. The SC dosed construct also shows preferential lymphatic transport, with over 50% of the bioavailable dose recovered in thoracic lymph. Increases in dose (up to 400 mg/kg) are well tolerated after SC administration and studies in a model of disseminated lymphoma in mice show that high dose SC treatment outperforms IV administration using doses that lead to similar total plasma exposure (lower peak concentrations but extended exposure after SC). These data show that the DEP® dendrimer can act as a circulating drug depot accessing both the lymphatic and blood circulatory systems. SC administration improves lymphatic exposure and facilitates higher dose administration due to improved tolerability. Higher dose SC administration also results in improved efficacy, suggesting that drug delivery systems that access both plasma and lymph hold significant potential for the treatment of haematological cancers where lymphatic and extranodal dissemination are poor prognostic factors.
Subject(s)
Antineoplastic Agents , Dendrimers , Lymphoma , Animals , Dendrimers/chemistry , Injections, Subcutaneous , Lymph , Lymphatic System , Lymphoma/drug therapy , MiceABSTRACT
Hyperbranched polymers have many promising features for drug delivery, owing to their ease of synthesis, multiple functional group content, and potential for high drug loading with retention of solubility. Here we prepared hyperbranched N-(2-hydroxypropyl)methacrylamide (HPMA) polymers with a range of molar masses and particle sizes, and with attached dyes, radiolabel or the anticancer drug gemcitabine. Reversible addition-fragmentation chain transfer (RAFT) polymerisation enabled the synthesis of pHPMA polymers and a gemcitabine-comonomer functionalised pHPMA polymer pro-drug, with diameters of the polymer particles ranging from 7-40 nm. The non-drug loaded polymers were well-tolerated in cancer cell lines and macrophages, and were rapidly internalised in 2D cell culture and transported efficiently to the centre of dense pancreatic cancer 3D spheroids. The gemcitabine-loaded polymer pro-drug was found to be toxic both to 2D cultures of MIA PaCa-2 cells and also in reducing the volume of MIA PaCa-2 spheroids. The non-drug loaded polymers caused no short-term adverse effects in healthy mice following systemic injection, and derivatives of these polymers labelled with 89Zr-were tracked for their distribution in the organs of healthy and MIA PaCa-2 xenograft bearing Balb/c nude mice. Tumour accumulation, although variable across the samples, was highest in individual animals for the pHPMA polymer of â¼20 nm size, and accordingly a gemcitabine pHPMA polymer pro-drug of â¼18 nm diameter was evaluated for efficacy in the tumour-bearing animals. The efficacy of the pHPMA polymer pro-drug was very similar to that of free gemcitabine in terms of tumour growth retardation, and although there was a survival benefit after 70 days for the polymer pro-drug, there was no difference at day 80. These data suggest that while polymer pro-drugs of this type can be effective, better tumour targeting and enhanced in situ release remain as key obstacles to clinical translation even for relatively simple polymers such as pHPMA.
Subject(s)
Neoplasms , Prodrugs , Acrylamides , Animals , Cell Line , Cell Line, Tumor , Humans , Mice , Mice, Nude , PolymersABSTRACT
Gaining insight into the heterogeneity of nanoparticle drug distribution within tumors would improve both design and clinical translation of nanomedicines. There is little data showing the spatio-temporal behavior of nanomedicines in tissues as current methods are not able to provide a comprehensive view of the nanomedicine distribution, released drug or its effects in the context of a complex tissue microenvironment. Methods: A new experimental approach which integrates the molecular imaging and bioanalytical technologies MSI and IMC was developed to determine the biodistribution of total drug and drug metabolite delivered via PLA-PEG nanoparticles and to overlay this with imaging of the nanomedicine in the context of detailed tumor microenvironment markers. This was used to assess the nanomedicine AZD2811 in animals bearing three different pre-clinical PDX tumors. Results: This new approach delivered new insights into the nanoparticle/drug biodistribution. Mass spectrometry imaging was able to differentiate the tumor distribution of co-dosed deuterated non-nanoparticle-formulated free drug alongside the nanoparticle-formulated drug by directly visualizing both delivery approaches within the same animal or tissue. While the IV delivered free drug was uniformly distributed, the nanomedicine delivered drug was heterogeneous. By staining for multiple biomarkers of the tumor microenvironment on the same tumor sections using imaging mass cytometry, co-registering and integrating data from both imaging modalities it was possible to determine the features in regions with highest nanomedicine distribution. Nanomedicine delivered drug was associated with regions higher in macrophages, as well as more stromal regions of the tumor. Such a comparison of complementary molecular data allows delineation of drug abundance in individual cell types and in stroma. Conclusions: This multi-modal imaging solution offers researchers a better understanding of drug and nanocarrier distribution in complex tissues and enables data-driven drug carrier design.
Subject(s)
Nanoparticles , Neoplasms , Animals , Drug Carriers/therapeutic use , Drug Delivery Systems , Molecular Imaging , Nanomedicine/methods , Nanoparticles/chemistry , Neoplasms/diagnostic imaging , Neoplasms/drug therapy , Tissue Distribution , Tumor MicroenvironmentABSTRACT
Dietary lipids and some pharmaceutical lipid excipients can facilitate the targeted delivery of drugs to the intestinal lymphatics. Here, the feasibility of magnetic resonance imaging (MRI) for imaging lipid uptake into the intestinal lymphatics was assessed, shedding light on which lymph nodes can be targeted using this approach. Three healthy male volunteers were scanned at 3.0 T at baseline, 120, 180, 240, and 300 min post high-fat meal. A sagittal multi-slice image was acquired using a diffusion-weighted whole-body imaging sequence with background suppression (DWIBS) (pre inversion TI = 260 ms). Changes in area, major, and minor axis length were compared at each time point. Apparent diffusion coefficient (ADC) was calculated (b = 0 and 600 s/mm2) across eight slices. An average of 22 nodes could be visualised across all time points. ADC increased at 120 and 180 min compared to the baseline in all three participants by an average of 9.2% and 6.8%, respectively. In two participants, mean node area and major axis lengths increased at 120 and 180 min relative to the baseline. In conclusion, the method described shows potential for repeated lymph node measurements and the tracking of lipid uptake into the lymphatics. Further studies should focus on methodology optimisation in a larger cohort.
ABSTRACT
Imaging mass cytometry (IMC) offers the opportunity to image metal- and heavy halogen-containing xenobiotics in a highly multiplexed experiment with other immunochemistry-based reagents to distinguish uptake into different tissue structures or cell types. However, in practice, many xenobiotics are not amenable to this analysis, as any compound which is not bound to the tissue matrix will delocalize during aqueous sample-processing steps required for IMC analysis. Here, we present a strategy to perform IMC experiments on a water-soluble polysarcosine-modified dendrimer drug-delivery system (S-Dends). This strategy involves two consecutive imaging acquisitions on the same tissue section using the same instrumental platform, an initial laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MSI) experiment followed by tissue staining and a standard IMC experiment. We demonstrated that settings can be found for the initial ablation step that leave sufficient residual tissue for subsequent antibody staining and visualization. This workflow results in lateral resolution for the S-Dends of 2 µm followed by imaging of metal-tagged antibodies at 1 µm.
Subject(s)
Image Cytometry , Water , Drug Delivery Systems , Mass Spectrometry , Staining and LabelingABSTRACT
Dual Bcl-2/Bcl-xL inhibitors are expected to deliver therapeutic benefit in many haematological and solid malignancies, however, their use is limited by tolerability issues. AZD4320, a potent dual Bcl-2/Bcl-xL inhibitor, has shown good efficacy however had dose limiting cardiovascular toxicity in preclinical species, coupled with challenging physicochemical properties, which prevented its clinical development. Here, we describe the design and development of AZD0466, a drug-dendrimer conjugate, where AZD4320 is chemically conjugated to a PEGylated poly-lysine dendrimer. Mathematical modelling was employed to determine the optimal release rate of the drug from the dendrimer for maximal therapeutic index in terms of preclinical anti-tumour efficacy and cardiovascular tolerability. The optimised candidate is shown to be efficacious and better tolerated in preclinical models compared with AZD4320 alone. The AZD4320-dendrimer conjugate (AZD0466) identified, through mathematical modelling, has resulted in an improved therapeutic index and thus enabled progression of this promising dual Bcl-2/Bcl-xL inhibitor into clinical development.
Subject(s)
Antineoplastic Agents , Dendrimers , Neoplasms/drug therapy , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Dendrimers/chemical synthesis , Dendrimers/chemistry , Dendrimers/pharmacokinetics , Dendrimers/therapeutic use , Dogs , Female , Humans , Male , Mice , Mice, Inbred C57BL , Mice, SCID , Neoplasms/metabolism , Neoplasms/pathology , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Rats , Rats, Wistar , Therapeutic Index , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , bcl-X Protein/antagonists & inhibitorsABSTRACT
This paper describes the synthesis of star polymers designed for future drug-delivery applications. A generation-5 lysine dendrimer was used as a macroinitiator for the ring-opening polymerization of the sarcosine N-carboxyanhydride monomer to produce 32-arm star polymers with narrow molar mass distributions and desirable hydrodynamic size control. Fluorescent dye-labeled polymers were dosed in mice to measure plasma pharmacokinetics. Long circulation times were observed, representing ideal properties for biophysical targeting of tumors. In vivo efficacy of one of these star polymers conjugated to the therapeutic molecule SN-38 was evaluated in mice bearing SW620 xenografted tumors to demonstrate high antitumor activity and low body weight loss compared to the SN-38 prodrug irinotecan and this shows the potential of these delivery systems. As a further build, we demonstrated that these star polymers can be easily chain-end-functionalized with useful chemical moieties, giving opportunities for future receptor-targeting strategies. Finally, we describe the synthetic advantages of these star polymers that make them attractive from a pharmaceutical manufacturing perspective and report characterization of the polymers with a variety of techniques.
Subject(s)
Dendrimers , Pharmaceutical Preparations , Animals , Mice , Peptides , Polymers , Sarcosine/analogs & derivativesABSTRACT
Glioblastoma multiforme (GBM) is a grade IV astrocytoma, which is the most aggressive form of brain tumor. The standard of care for this disease includes surgery, radiotherapy and temozolomide (TMZ) chemotherapy. Poor accumulation of TMZ at the tumor site, tumor resistance to drug, and dose-limiting bone marrow toxicity eventually reduce the success of this treatment. Herein, we have encapsulated >500 drug molecules of TMZ into the biocompatible protein nanocage, apoferritin (AFt), using a "nanoreactor" method (AFt-TMZ). AFt is internalized by transferrin receptor 1-mediated endocytosis and is therefore able to facilitate cancer cell uptake and enhance drug efficacy. Following encapsulation, the protein cage retained its morphological integrity and surface charge; hence, its cellular recognition and uptake are not affected by the presence of this cargo. Additional benefits of AFt include maintenance of TMZ stability at pH 5.5 and drug release under acidic pH conditions, encountered in lysosomal compartments. MTT assays revealed that the encapsulated agents displayed significantly increased antitumor activity in U373V (vector control) and, remarkably, the isogenic U373M (MGMT expressing TMZ-resistant) GBM cell lines, with GI50 values <1.5 µM for AFt-TMZ, compared to 35 and 376 µM for unencapsulated TMZ against U373V and U373M, respectively. The enhanced potency of AFt-TMZ was further substantiated by clonogenic assays. Potentiated G2/M cell cycle arrest following exposure of cells to AFt-TMZ indicated an enhanced DNA damage burden. Indeed, increased O6-methylguanine (O6-MeG) adducts in cells exposed to AFt-TMZ and subsequent generation of γH2AX foci support the hypothesis that AFt significantly enhances the delivery of TMZ to cancer cells in vitro, overwhelming the direct O6-MeG repair conferred by MGMT. We have additionally encapsulated >500 molecules of the N3-propargyl imidazotetrazine analog (N3P), developed to combat TMZ resistance, and demonstrated significantly enhanced activity of AFt-N3P against GBM and colorectal carcinoma cell lines. These studies support the use of AFt as a promising nanodelivery system for targeted delivery, lysosomal drug release, and enhanced imidazotetrazine potency for treatment of GBM and wider-spectrum malignancies.
Subject(s)
Antineoplastic Agents, Alkylating , Apoferritins/chemistry , Brain Neoplasms/metabolism , Nanostructures/chemistry , Temozolomide , Antineoplastic Agents, Alkylating/chemistry , Antineoplastic Agents, Alkylating/pharmacokinetics , Antineoplastic Agents, Alkylating/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Delivery Systems , Glioblastoma/metabolism , Humans , Temozolomide/analogs & derivatives , Temozolomide/chemistry , Temozolomide/pharmacokinetics , Temozolomide/pharmacologyABSTRACT
To improve the clinical translation of anti-cancer nanomedicines, it is necessary to begin building specific insights into the broad concept of the Enhanced Permeability and Retention (EPR) effect, using detailed investigations of the accumulation, distribution and retention of nanomedicines in solid tumors. Nanomedicine accumulation in preclinical tumors has been extensively studied; however, treatment efficacy will be heavily influenced by both the quantity of drug-loaded nanomedicines reaching the tumor as well as their spatial distribution throughout the tumor. It remains a challenge to image the heterogeneity of nanomedicine distribution in 3 dimensions within solid tumors with a high degree of spatial resolution using standard imaging approaches. Methods: To achieve this, an ex vivo micro computed tomography (µCT) imaging approach was developed to visualize the intratumoral distribution of contrast agent-loaded PEGylated liposomes. Using this semi-quantitative method, whole 3-dimensional (3D) tumor liposome distribution was determined with 17 µm resolution in a phenotypically diverse panel of four preclinical xenograft and patient-derived explant (PDX) tumor models. Results: High-resolution ex vivo µCT imaging revealed striking differences in liposome distribution within tumors in four models with different vascular patterns and densities, stromal contents, and microenvironment morphologies. Following intravenous dosing, the model with the highest density of pericyte-supported vessels showed the greatest liposome accumulation, while the model with vessels present in regions of high α-smooth muscle actin (αSMA) content presented with a large proportion of the liposomes at depths beyond the tumor periphery. The two models with an unsupported vascular network demonstrated a more restricted pattern of liposome distribution. Conclusion: Taken together, vessel distribution and support (the latter indicative of functionality) appear to be key factors determining the accumulation and distribution pattern of liposomes in tumors. Our findings demonstrate that high-resolution 3D visualization of nanomedicine distribution is a useful tool for preclinical nanomedicine research, providing valuable insights into the influence of the tumor vasculature and microenvironment on nanomedicine localization.
Subject(s)
Contrast Media/pharmacology , Imaging, Three-Dimensional/methods , Liposomes/chemistry , Nanomedicine/methods , Nanoparticles/administration & dosage , Neoplasms/diagnostic imaging , X-Ray Microtomography/methods , Animals , Cell Line, Tumor , Contrast Media/chemistry , Humans , Male , Mice , Mice, SCID , Nanoparticles/chemistry , Neoplasms/drug therapy , Neoplasms/pathology , Permeability , Tumor Microenvironment , Xenograft Model Antitumor AssaysABSTRACT
Colorectal cancer remains a significant cause of morbidity and mortality worldwide. Half of all patients develop liver metastases, presenting unique challenges for their treatment. The shortcomings of conventional chemotherapy has encouraged the use of nanomedicines; the application of nanotechnology in the diagnosis and treatment of disease. In spite of technological improvements in nanotechnology, the complexity of biological systems hinders the prospect of nanomedicines being applied in cancer therapy at the present time. This review highlights current biological barriers and discusses aspects of tumor biology together with the physicochemical features of the nanocarrier, that need to be considered in order to develop effective nanotherapeutics for colorectal cancer patients with liver metastases. It becomes clear that incorporating an interdisciplinary approach when developing nanomedicines should assure appropriate disease-driven design and that this will form a critical step in improving their clinical translation. This article is characterized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.
Subject(s)
Colorectal Neoplasms , Drug Delivery Systems , Liver Neoplasms , Nanomedicine , Nanoparticles , Animals , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Humans , Liver Neoplasms/drug therapy , Liver Neoplasms/secondary , MiceABSTRACT
Lipid nanoparticles have great potential for delivering nucleic-acid-based therapeutics, but low efficiency limits their broad clinical translation. Differences in transfection capacity between in vitro models used for nanoparticle pre-clinical testing are poorly understood. To address this, using a clinically relevant lipid nanoparticle (LNP) delivering mRNA, we highlight specific endosomal characteristics in in vitro tumor models that impact protein expression. A 30-cell line LNP-mRNA transfection screen identified three cell lines having low, medium, and high transfection that correlated with protein expression when they were analyzed in tumor models. Endocytic profiling of these cell lines identified major differences in endolysosomal morphology, localization, endocytic uptake, trafficking, recycling, and endolysosomal pH, identified using a novel pH probe. High-transfecting cells showed rapid LNP uptake and trafficking through an organized endocytic pathway to lysosomes or rapid exocytosis. Low-transfecting cells demonstrated slower endosomal LNP trafficking to lysosomes and defective endocytic organization and acidification. Our data establish that efficient LNP-mRNA transfection relies on an early and narrow endosomal escape window prior to lysosomal sequestration and/or exocytosis. Endocytic profiling should form an important pre-clinical evaluation step for nucleic acid delivery systems to inform model selection and guide delivery-system design for improved clinical translation.
Subject(s)
Gene Expression , Lipids/chemistry , Nanoparticles , RNA, Messenger/genetics , Transfection , Cell Line, Tumor , Endocytosis , Endosomes/metabolism , Flow Cytometry , Fluorescent Antibody Technique , Genes, Reporter , Humans , Hydrogen-Ion Concentration , Lysosomes/metabolism , Nanoparticles/chemistry , RNA, Messenger/administration & dosage , Transfection/methodsABSTRACT
We report on the synthesis of four poly(2-methyl-2-oxazoline) modified lysine dendrimers with different residual groups or modifications on the dendrimer core, including: amino groups (positive charge), carboxyl groups (negative charge), and two drug molecules, one of which has a high log P. We looked at the in vivo distribution amongst three main liver cell types: hepatocytes, liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs) and found differences in cell distribution and uptake concentrations dependent on these residual groups. In particular, the amino-functional polymer showed greater uptake by the hepatocytes whilst the carboxyl-functionalised polymer exhibited greater uptake by KCs and LSECs. These findings provide insight into which professional scavenger cells of the liver remove these types of nanoparticles from the bloodstream and we describe some of the design criteria to consider when creating novel drug delivery systems.
Subject(s)
Dendrimers/chemistry , Liver/metabolism , Lysine/chemistry , Lysine/metabolism , Polyamines/chemistry , Administration, Intravenous , Animals , Biological Transport , Female , Hydrophobic and Hydrophilic Interactions , Lysine/administration & dosage , Lysine/pharmacokinetics , Mice , Rhodamines/chemistry , Tissue DistributionABSTRACT
The development of delivery systems capable of tumor targeting represents a promising strategy to overcome issues related to nonspecific effects of conventional anticancer therapies. Currently, one of the most investigated agents for cancer targeting is hyaluronic acid (HA), since its receptor, CD44, is overexpressed in many cancers. However, most of the studies on CD44/HA interaction have been so far performed in cell-free or genetically modified systems, thus leaving some uncertainty regarding which cell-related factors influence HA binding and internalization (collectively called "uptake") into CD44-expressing cells. To address this, the expression of CD44 (both standard and variants, designated CD44s and CD44v, respectively) was evaluated in human dermal fibroblasts (HDFs) and a large panel of cancer cell lines, including breast, prostate, head and neck, pancreatic, ovarian, colorectal, thyroid, and endometrial cancers. Results showed that CD44 isoform profiles and expression levels vary across the cancer cell lines and HDF and are not consistent within the cell origin. Using composite information of CD44 expression, HA binding, and internalization, we found that the expression of CD44v can negatively influence the uptake of HA, and, instead, when cells primarily expressed CD44s, a positive correlation was observed between expression and uptake. In other words, CD44shigh cells bound and internalized more HA compared to CD44slow cells. Moreover, CD44shigh HDFs were less efficient in uptaking HA compared to CD44shigh cancer cells. The experiments described here are the first step toward understanding the interplay between CD44 expression, its functionality, and the underlying mechanism(s) for HA uptake. The results show that factors other than the amount of CD44 receptor can play a role in the interaction with HA, and this represents an important advance with respect to the design of HA-based carriers and the selection of tumors to treat according to their CD44 expression profile.
Subject(s)
Hyaluronan Receptors/metabolism , Hyaluronic Acid/chemistry , Hyaluronic Acid/therapeutic use , Cell Line, Tumor , Drug Delivery Systems/methods , Flow Cytometry , Humans , ImmunohistochemistryABSTRACT
Glioblastoma multiforme is the most common and lethal brain tumour-type. The current standard of care includes Temozolomide (TMZ) chemotherapy. However, inherent and acquired resistance to TMZ thwart successful treatment. The direct repair protein methylguanine DNA methyltransferase (MGMT) removes the cytotoxic O6-methylguanine (O6-MeG) lesion delivered by TMZ and so its expression by tumours confers TMZ-resistance. DNA mismatch repair (MMR) is essential to process O6-MeG adducts and MMR-deficiency leads to tolerance of lesions, resistance to TMZ and further DNA mutations. In this article, two strategies to overcome TMZ resistance are discussed: (1) synthesis of imidazotetrazine analogues - designed to retain activity in the presence of MGMT or loss of MMR; (2) preparation of imidazotetrazine-nanoparticles to deliver TMZ preferably to the brain and tumour site. Our promising results encourage belief in a future where better prognoses exist for patients diagnosed with this devastating disease.
ABSTRACT
A small library of thermoresponsive amphiphilic copolymers based on polylactide-block-poly((2-(2-methoxyethoxy)ethyl methacrylate)-co-(oligoethylene glycol methacrylate)) (PLA-b-P(DEGMA)-co-(OEGMA)), was synthesised by copper-mediated controlled radical polymerisation (CRP) with increasing ratios of OEGMA : DEGMA. These polymers were combined in two ways to form nanoparticles with controllable thermal transition temperatures as measured by particle aggregation. The first technique involved the blending of two (PLA-b-P(DEGMA)-co-(OEGMA)) polymers together prior to assembling nanoparticles (NPs). The second method involved mixing pre-formed nanoparticles of single (PLA-b-P(DEGMA)-co-(OEGMA)) polymers. The observed critical aggregation temperature Tt did not change in a linear relationship with the ratios of each copolymer either in the nanoparticles blended from different copolymers or in the mixtures of pre-formed nanoparticles. However, where co-polymer mixtures were based on (OEG)9MA ratios within 5-10 mole%, a linear relationship between (OEG)9MA composition in the blends and Tt was obtained. The data suggest that OEGMA-based copolymers are tunable over a wide temperature range given suitable co-monomer content in the linear polymers or nanoparticles. Moreover, the thermal transitions of the nanoparticles were reversible and repeatable, with the cloud point curves being essentially invariant across at least three heating and cooling cycles, and a selected nanoparticle formulation was found to be readily endocytosed in representative cancer cells and fibroblasts.
Subject(s)
Biocompatible Materials/chemistry , Nanoparticles/chemistry , Polymers/chemistry , Temperature , Biocompatible Materials/toxicity , Humans , MCF-7 Cells , Polymers/toxicityABSTRACT
Irinotecan is used clinically for the treatment of colorectal cancer; however, its utility is limited by its narrow therapeutic index. We describe the use of a generation 5 l-lysine dendrimer that has been part-modified with a polyoxazoline as a drug delivery vehicle for improving the therapeutic index of SN-38, the active metabolite of irinotecan. By conjugating SN-38 to the dendrimer via different linker technologies we sought to vary the release rate of the drug to generate diverse pharmacokinetic profiles. Three conjugates with plasma release half-lives of 2.5h, 21h, and 72h were tested for efficacy and toxicity using a mouse SW620 xenograft model. In this model, the linker with a plasma release half-life of 21h achieved sustained SN-38 exposure in blood, above the target concentration. Control over the release rate of the drug from the linker, combined with prolonged circulation of the dendrimer, enabled administration of an efficacious dose of SN-38, achieving significant regression of the SW620 tumours. The conjugates with 2.5 and 72h release half-lives did not achieve an anti-tumour effect. Intraperitoneal dosing of the clinically used prodrug irinotecan produces high initial and local concentrations of SN-38, which are associated with gastrointestinal toxicity. Administration of the 21h release dendrimer conjugate did not produce a high initial Cmax of SN-38. Consequently, a marked reduction in gastrointestinal toxicity was observed relative to irinotecan treatment. Additional studies investigating the dose concentrations and dose scheduling showed that a weekly dosing schedule of 4mg SN-38/kg was the most efficacious regimen. After 4 doses at weekly intervals, the survival period of the mice extended beyond 70 days following the final dose. These extensive studies have allowed us to identify a linker, dose and dosing regimen for SN-38 conjugated to polyoxazoline-modified dendrimer that maximised efficacy and minimised adverse side effects.