Quantitative Evaluation of Dendritic Nanoparticles in Mice: Biodistribution Dynamics and Downstream Tumor Efficacy Outcomes.

A physiologically based pharmacokinetic model was developed to describe the tissue distribution kinetics of a dendritic nanoparticle and its conjugated active pharmaceutical ingredient (API) in plasma, liver, spleen, and tumors. Tumor growth data from MV-4-11 tumor-bearing mice were incorporated to investigate the exposure/efficacy relationship. The nanoparticle demonstrated improved antitumor activity compared to the conventional API formulation, owing to the extended released API concentrations at the site of action. Model simulations further enabled the identification of critical parameters that influence API exposure in tumors and downstream efficacy outcomes upon nanoparticle administration. The model was utilized to explore a range of dosing schedules and their effect on tumor growth kinetics, demonstrating the improved antitumor activity of nanoparticles with less frequent dosing compared to the same dose of naked APIs in conventional formulations.

Method to Investigate the Distribution of Water-Soluble Drug-Delivery Systems in Fresh Frozen Tissues Using Imaging Mass Cytometry.

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.

Synthesis and Characterization of Dendrimer-Based Polysarcosine Star Polymers: Well-Defined, Versatile Platforms Designed for Drug-Delivery Applications.

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.

Endocytic Profiling of Cancer Cell Models Reveals Critical Factors Influencing LNP-Mediated mRNA Delivery and Protein Expression.

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.

Penetration and Uptake of Nanoparticles in 3D Tumor Spheroids.

Animal models are effective for assessing tumor localization of nanosystems but difficult to use for studying penetration beyond the vasculature. Here, we have used well-characterized HCT116 colorectal cancer spheroids to study the effect of nanoparticle (NP) physicochemical properties on penetration and uptake. Incubation of spheroids with Hoechst 33342 resulted in a dye gradient, which facilitated discrimination between the populations of cells in the core and at the periphery of spheroids by flow cytometry. This approach was used to compare doxorubicin and liposomal doxorubicin (Caelyx) and a range of model poly(styrene) nanoparticles of different sizes (30 nm, 50 nm, 100 nm) and with different surface chemistries (50 nm uniform plain, carboxylated, aminated and a range of NPs and polyethylene glycol modified NPs prepared from a promising new functionalized biodegradable polymer (poly(glycerol-adipate), PGA). Unmodified poly(styrene) nanoparticles (30 nm/50 nm) were able to penetrate to the core of HCT116 spheroids more efficiently than larger poly(styrene) nanoparticles (100 nm). Surprisingly, penetration of 30 and 50 nm particles was as good as clinically relevant doxorubicin concentrations. However, penetration was reduced with higher surface charge. PGA NPs of 100 nm showed similar penetration into spheroids as 50 nm poly(styrene) nanoparticles, which may be related to polymer flexibility. PEG surface modification of polymeric particles significantly improved penetration into the spheroid core. The new model combining the use of spheroids Hoechst staining and flow cytometry was a useful model for assessing NP penetration and gives useful insights into the effects of NPs' physical properties when designing nanomedicines.

Evaluating the Efficiency of Hyaluronic Acid for Tumor Targeting via CD44.

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.

Enhanced Intraliposomal Metallic Nanoparticle Payload Capacity Using Microfluidic-Assisted Self-Assembly.

Hybrids composed of liposomes (L) and metallic nanoparticles (NPs) hold great potential for imaging and drug delivery purposes. However, the efficient incorporation of metallic NPs into liposomes using conventional methodologies has so far proved to be challenging. In this study, we report the fabrication of hybrids of liposomes and hydrophobic gold NPs of size 2-4 nm (Au) using a microfluidic-assisted self-assembly process. The incorporation of increasing amounts of AuNPs into liposomes was examined using microfluidics and compared to L-AuNP hybrids prepared by the reverse-phase evaporation method. Our microfluidics strategy produced L-AuNP hybrids with a homogeneous size distribution, a smaller polydispersity index, and a threefold increase in loading efficiency when compared to those hybrids prepared using the reverse-phase method of production. Quantification of the loading efficiency was determined by ultraviolet spectroscopy, inductively coupled plasma mass spectroscopy, and centrifugal field flow fractionation, and qualitative validation was confirmed by transmission electron microscopy. The higher loading of gold NPs into the liposomes achieved using microfluidics produced a slightly thicker and more rigid bilayer as determined with small-angle neutron scattering. These observations were confirmed using fluorescent anisotropy and atomic force microscopy. Structural characterization of the liposomal-NP hybrids with cryo-electron microscopy revealed the coexistence of membrane-embedded and interdigitated NP-rich domains, suggesting AuNP incorporation through hydrophobic interactions. The microfluidic technique that we describe in this study allows for the automated production of monodisperse liposomal-NP hybrids with high loading capacity, highlighting the utility of microfluidics to improve the payload of metallic NPs within liposomes, thereby enhancing their application for imaging and drug delivery.

Chitosan/Hyaluronic Acid Nanoparticles: Rational Design Revisited for RNA Delivery.

Chitosan/hyaluronic acid (HA) nanoparticles can be used to deliver an RNA/DNA cargo to cells overexpressing HA receptors such as CD44. For these systems, unequivocal links have not been established yet between chitosan macromolecular (molecular weight; degree of deacetylation, i.e., charge density) and nanoparticle variables (complexation strength, i.e., stability; nucleic acid protection; internalization rate) on one hand, and transfection efficiency on the other hand. Here, we have focused on the role of avidity on transfection efficiency in the CD44-expressing HCT-116 as a cellular model; we have employed two differently sized payloads (a large luciferase-encoding mRNA and a much smaller anti-Luc siRNA), and a small library of chitosans (variable molecular weight and degree of deactylation). The RNA avidity for chitosan showed-as expected-an inverse relationship: higher avidity-higher polyplex stability-lower transfection efficiency. The avidity of chitosan for RNA appears to lead to opposite effects: higher avidity-higher polyplex stability but also higher transfection efficiency. Surprisingly, the best transfecting particles were those with the lowest propensity for RNA release, although this might be a misleading relationship: for example, the same macromolecular parameters that increase avidity can also boost chitosan's endosomolytic activity, with a strong enhancement in transfection. The performance of these nonviral vectors appears therefore difficult to predict simply on the basis of carrier- or payload-related variables, and a more holistic consideration of the journey of the nanoparticle, from cell uptake to cytosolic bioavailability of payload, is needed. It is also noteworthy that the nanoparticles used in this study showed optimal performance under slightly acidic conditions (pH 6.4), which is promising for applications in a tumoral extracellular environment. It is also worth pointing out that under these conditions we have for the first time successfully delivered mRNA with chitosan/HA nanoparticles.

Control of aggregation temperatures in mixed and blended cytocompatible thermoresponsive block co-polymer nanoparticles.

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.

Insights into low molecular mass organic gelators: a focus on drug delivery and tissue engineering applications.

In recent years low molecular mass organic gelators (LMOGs) have gained increasing interest as an alternative biomaterial to polymer derived gels, with potential applications in drug delivery and tissue engineering. LMOGs are small organic molecules which self-assemble in water or organic solvents forming a 3D network that entraps the liquid phase resulting in gel formation. In this review, we report the classification of LMOGs into hydrogelators and gelators of organic solvents and we discuss the techniques commonly used to characterise the gels of these gelators with particular reference to specific applications of LMOGs in drug delivery and tissue engineering.

Free drug and ROS-responsive nanoparticle delivery of synergistic doxorubicin and olaparib combinations to triple negative breast cancer models.

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.

Impact of the physical-chemical properties of poly(lactic acid)-poly(ethylene glycol) polymeric nanoparticles on biodistribution.

Nanoparticle (NP) formulations are inherently polydisperse making their structural characterization and justification of specifications complex. It is essential, however, to gain an understanding of the physico-chemical properties that drive performance in vivo. To elucidate these properties, drug-containing poly(lactic acid) (PLA)-poly(ethylene glycol) (PEG) block polymeric NP formulations (or PNPs) were sub-divided into discrete size fractions and analyzed using a combination of advanced techniques, namely cryogenic transmission electron microscopy, small-angle neutron and X-ray scattering, nuclear magnetic resonance, and hard-energy X-ray photoelectron spectroscopy. Together, these techniques revealed a uniquely detailed picture of PNP size, surface structure, internal molecular architecture and the preferred site(s) of incorporation of the hydrophobic drug, AZD5991, properties which cannot be accessed via conventional characterization methodologies. Within the PNP size distribution, it was shown that the smallest PNPs contained significantly less drug than their larger sized counterparts, reducing overall drug loading, while PNP molecular architecture was critical in understanding the nature of in vitro drug release. The effect of PNP size and structure on drug biodistribution was determined by administrating selected PNP size fractions to mice, with the smaller sized NP fractions increasing the total drug-plasma concentration area under the curve and reducing drug concentrations in liver and spleen, due to greater avoidance of the reticuloendothelial system. In contrast, administration of unfractionated PNPs, containing a large population of NPs with extremely low drug load, did not significantly impact the drug's pharmacokinetic behavior - a significant result for nanomedicine development where a uniform formulation is usually an important driver. We also demonstrate how, in this study, it is not practicable to validate the bioanalytical methodology for drug released in vivo due to the NP formulation properties, a process which is applicable for most small molecule-releasing nanomedicines. In conclusion, this work details a strategy for determining the effect of formulation variability on in vivo performance, thereby informing the translation of PNPs, and other NPs, from the laboratory to the clinic.

Histopathological biomarkers for predicting the tumour accumulation of nanomedicines.

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.

Precise and systematic end group chemistry modifications on PAMAM and poly(l-lysine) dendrimers to improve cytosolic delivery of mRNA.

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.

In depth characterization of physicochemical critical quality attributes of a clinical drug-dendrimer conjugate.

A deep and detailed understanding of drug-dendrimer conjugates key properties is needed to define the critical quality attributes that affect drug product performance. The characterization must be executed both in the formulation media and in biological matrices. This, nevertheless, is challenging on account of a very limited number of suitable, established methods for characterizing the physicochemical properties, stability, and interaction with biological environment of complex drug-dendrimer conjugates. In order to fully characterize AZD0466, a drug-dendrimer conjugate currently under clinical development by AstraZeneca, a collaboration was initiated with the European Nanomedicine Characterisation Laboratory to deploy a state-of-the-art multi-step approach to measure physicochemical properties. An incremental complexity characterization approach was applied to two batches of AZD0466 and the corresponding dendrimer not carrying any drug, SPL-8984. Thus, the aim of this work is to guide in depth characterization efforts in the analysis of drug-dendrimer conjugates. Additionally, it serves to highlight the importance of using the adequate complementary techniques to measure physical and chemical stability in both simple and biological media, to drive a complex drug-dendrimer conjugate product from discovery to clinical development.

A View on Drug Development for Cancer Prevention.

Despite some notable successes, there are still relatively few agents approved for cancer prevention. Here we review progress thus far in the development of medicines for cancer prevention, and we outline some key concepts that could further enable or accelerate drug development for cancer prevention in the future. These are summarized under six key themes: (i) unmet clinical need, (ii) patient identification, (iii) risk stratification, (iv) pharmacological intervention, (v) clinical trials, and (vi) health care policy. These concepts, if successfully realized, may help to increase the number of medicines available for cancer prevention. SIGNIFICANCE: The huge potential public health benefits of preventing cancer, combined with recent advances in the availability of novel early detection technologies and new treatment modalities, has caused us to revisit the opportunities and challenges associated with developing medicines to prevent cancer. Here we review progress in the field of developing medicines to prevent cancer to date, and we present a series of ideas that might help in the development of more medicines to prevent cancer in the future.

The Global Characterisation of a Drug-Dendrimer Conjugate - PEGylated poly-lysine Dendrimer.

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.

Nucleic Acid-Loaded Lipid Nanoparticle Interactions with Model Endosomal Membranes.

Lipid nanoparticles (LNPs) are important delivery systems for RNA-based therapeutics, yet the mechanism of their interaction with endosomal membranes remains unclear. Here, the interactions of nucleic acid-loaded LNPs that contain an ionizable lipid with models of the early and late endosomal membranes are studied, for the first time, using different reflectometry techniques. Novel insight is provided with respect to the subphase pH, the stage of the endosome, and the nature of the nucleic acid cargo. It is found that the insertion of lipids from the LNPs into the model membrane is greatest at pH 6.5 and 5.5, whereas at higher pH, lipid insertion is suppressed with evidence instead for the binding of intact LNPs, demonstrating the importance of the pH in the fusion of LNPs undergoing the endosomal pathway. Furthermore, and independently of the pH, the effect of the early- versus late-stage endosomal models is minimal, suggesting that the increased fluidity and anionic nature of the late endosome has little effect on the extent of LNP interaction. Last, there is greater nucleic acid delivery from LNPs containing mRNA than Poly(A), indicating that the extent of interaction can be tuned according to the nature of the nucleic acid cargo. Such new information on the relative impact of factors influencing nucleic acid delivery by LNP interactions with endosomal membranes is important in the design and tuning of vehicles with improved nucleic acid delivery capacities.

Distribution of a highly lipophilic drug cannabidiol into different lymph nodes following oral administration in lipidic vehicle.

Efficient delivery of highly lipophilic drugs or prodrugs to the mesenteric lymph nodes (MLN) can be achieved following oral administration with lipids. However, it remains unclear which specific MLN can be targeted and to what extent. Moreover, the efficiency of drug delivery to the retroperitoneal lymph nodes (RPLN) has not been assessed. The aim of this study was to assess the distribution of a highly lipophilic model drug cannabidiol (CBD), known to undergo intestinal lymphatic transport following administration with lipids, into specific MLN and RPLN in rats at various time-points post dosing. In vivo studies showed that at 2 h following administration, significantly higher concentrations of CBD were present in the region second from the apex of the MLN chain. From 3 h following administration, concentrations in all MLN were similar. CBD was also found at substantial levels in RPLN. This study demonstrates that drug concentrations in specific MLN are different, at least at the peak of the absorption process. Moreover, in addition to the MLN, the RPLN may also be targeted by oral route of administration, which may have further implications for treatment of a range of diseases.

Multi-modal molecular imaging maps the correlation between tumor microenvironments and nanomedicine distribution.

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.