Quantitative imaging of intracellular nanoparticle exposure enables prediction of nanotherapeutic efficacy.

Nanoparticle internalisation is crucial for the precise delivery of drug/genes to its intracellular targets. Conventional quantification strategies can provide the overall profiling of nanoparticle biodistribution, but fail to unambiguously differentiate the intracellularly bioavailable particles from those in tumour intravascular and extracellular microenvironment. Herein, we develop a binary ratiometric nanoreporter (BiRN) that can specifically convert subtle pH variations involved in the endocytic events into digitised signal output, enabling the accurately quantifying of cellular internalisation without introducing extracellular contributions. Using BiRN technology, we find only 10.7-28.2% of accumulated nanoparticles are internalised into intracellular compartments with high heterogeneity within and between different tumour types. We demonstrate the therapeutic responses of nanomedicines are successfully predicted based on intracellular nanoparticle exposure rather than the overall accumulation in tumour mass. This nonlinear optical nanotechnology offers a valuable imaging tool to evaluate the tumour targeting of new nanomedicines and stratify patients for personalised cancer therapy.

Exosomes Harnessed as Nanocarriers for Cancer Therapy - Current Status and Potential for Future Clinical Applications.

Exosomes are nano structured (50-90 nm) vesicles that originate from endosomal compartment of eukaryotic cells and are secreted into extracellular matrix. In recent years, there has been increased interest in exploring exosomes for diagnostic and therapeutic applications. Like many other diseases, e.g., neurodegenerative disorders, autoimmune diseases exosomes have a considerable significance in cancer too. Exosomes are known to prevail in large numbers and carry unique cargos in different types of cancers and thus are proving as versatile entities in understanding their biology of cancers and utilized as efficient diagnostic biomarkers in identification of cancer type. In addition to diagnostic applications, there has been an increased interest in recent years to exploit exosomes as carriers for delivery of therapeutic agents to target sites as well. This is indebted to their exceptional non-immunogenic and biomimetic properties that prompted researchers to use exosomes as carriers for delivery of therapeutic agents, e.g., drugs, genes and peptides. Exosomes also circumvent many drawbacks associated with other lipid or polymeric nanocarriers, e.g., low circulation time, lipid toxicities, long term stability, etc. However, in spite of many favorable aspects of exosome based therapy, there have been a number of challenges too. This review will focus on the current status of the exosome based drug therapy for cancer, the challenges faced and its potential for future clinical use.

Visualization of the distribution of nanoparticle-formulated AZD2811 in mouse tumor model using matrix-assisted laser desorption ionization mass spectrometry imaging.

Penetration of nanoparticles into viable tumor regions is essential for an effective response. Mass spectrometry imaging (MSI) is a novel method for evaluating the intratumoral pharmacokinetics (PK) of a drug in terms of spatial distribution. The application of MSI for analysis of nanomedicine PK remains in its infancy. In this study, we evaluated the applicability of MALDI-MSI for nanoparticle-formulated drug visualization in tumors and biopsies, with an aim toward future application in clinical nanomedicine research. We established an analytic method for the free drug (AZD2811) and then applied it to visualize nanoparticle-formulated AZD2811. MSI analysis demonstrated heterogeneous intratumoral drug distribution in three xenograft tumors. The intensity of MSI signals correlated well with total drug concentration in tumors, indicating that drug distribution can be monitored quantitatively. Analysis of tumor biopsies indicated that MSI is applicable for analyzing the distribution of nanoparticle-formulated drugs in tumor biopsies, suggesting clinical applicability.

MALDI-MS Imaging Analysis of Noninflammatory Type III Rotaxane Dendrimers.

Rotaxane dendrimers with hyperbranched macromolecular interlocked structures and size modulation capacity demonstrate drug binding and release ability upon external stimuli. Mass spectrometry imaging (MSI) can offer the high-throughput screening of endogenous/exogenous compounds. Herein, we reported a novel method to display the in situ spatial distribution of label-free monodispersed type III rotaxane dendrimers (RDs) G1 (first generation, size ∼1.5 nm) and G2 (second generation, size ∼5 nm) that were explored as potential drug vehicles in spleen tissue by using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-MSI). Experimental results indicated that the trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB) matrix exhibited the best performance for monodispersed type III RDs G1 and G2. The optimized method was successfully applied to map the in vivo spatial distribution of type III RDs G1 and G2 in the spleen from intraperitoneally injected mice. The MALDI-MSI images revealed that RDs G1 and G2 were relatively stable in the spleen within 24 h after administration. It was found that the identified type III RDs G1 and G2 penetrated through the tunica serosa and were predominantly localized in red pulp regions of spleens. They were also mapped in a marginal zone of spleens simultaneously. There was almost no toxicity of type III RDs G1 and G2 to mice spleens from the H&E results. Furthermore, the type III RDs did not induce the expression of inflammatory cytokines from peripheral blood mononuclear cells (PBMCs) or THP-1 monocytes. The MSI analysis not only demonstrated its ability to image select rotaxane dendrimers in a rapid and efficient manner but also provided tremendous assistance on the applications of the further treatment of cancerous tissue as safe drug carriers. Furthermore, the new strategy demonstrated in this study could be applied on other label-free mechanically interlocked molecules, molecular machines, and macromolecules, which opened a new path to evaluate the toxicological and pharmacokinetic characteristics of these novel materials at the suborgan level.

Characterization of chemical profiles of pH-sensitive cleavable D-gluconhydroximo-1, 5-lactam hydrolysates by LC-MS: A potential agent for promoting tumor-targeted drug delivery.

Currently, controllable linker cleavage at the target site will facilitate the clinical treatment of cancer. Dual-functional prodrugs in combination of carbohydrate as targeting group and pH-sensitive cleavable linker are desired in clinical development. Here, a qualified structure of N-phenylcarbamate-d-gluconhydroximo-1,5-lactam was employed and proved to be a potential candidate prodrug in the drug design. To proof this concept, the possible mechanism of Beckmann rearrangement and the degraded products were confirmed by HPLC and LC-MS under the acid condition mimic lysosome. Hence, the strategy of d-gluconhydroximo-1,5-lactam as a prodrug carrier fabricated with interested drugs will provide a great potential approach for chemotherapy.

Mass Spectrometric Detection and Characterization of Metabolites of Gemini Surfactants Used as Gene Delivery Vectors.

Gemini surfactants are a class of lipid molecules that have been successfully used in vitro and in vivo as nonviral gene delivery vectors. However, the biological fate of gemini surfactants has not been well investigated. In particular, the metabolism of gemini surfactants after they enter cells as gene delivery vehicles is unknown. In this work, we used a high-resolution quadrupole-Orbitrap mass spectrometry (Q-Exactive) instrument to detect the metabolites of three model gemini surfactants, namely, (a) unsubstituted (16-3-16), (b) with pyridinium head groups (16(Py)-S-2-S-16(Py)), and (c) substituted with a glycyl-lysine di-peptide (16-7N(GK)-16). The metabolites were characterized, and structures were proposed, based on accurate masses and characteristic product ions. The metabolism of the three gemini surfactants was very different as 16-3-16 was not metabolized in PAM 212 cells, whereas 16(Py)-S-2-S-16(Py) was metabolized primarily via phase I reactions, including oxidation and dealkylation, producing metabolites that could be linked to its observed high toxicity. The third gemini surfactant 16-7N(GK)-16 was metabolized mainly via phase II reactions, including methylation, acetylation, glucose conjugation, palmityl conjugation, and stearyl conjugation. The metabolism of gemini surfactants provides insight for future directions in the design and development of more effective gemini surfactants with lower toxicity. The reported approach can also be applied to study the metabolism of other structurally related gemini surfactants.

Probing the Assembly of HDL Mimetic, Drug Carrying Nanoparticles Using Intrinsic Fluorescence.

Reconstituted high-density lipoprotein (HDL) containing apolipoprotein A-I (Apo A-I) mimics the structure and function of endogenous (human plasma) HDL due to its function and potential therapeutic utility in atherosclerosis, cancer, neurodegenerative diseases, and inflammatory diseases. Recently, a new class of HDL mimetics has emerged, involving peptides with amino acid sequences that simulate the the primary structure of the amphipathic alpha helices within the Apo A-I protein. The findings reported in this communication were obtained using a similar amphiphilic peptide (modified via conjugation of a myristic acid residue at the amino terminal aspartic acid) that self-assembles (by itself) into nanoparticles while retaining the key features of endogenous HDL. The studies presented here involve the macromolecular assembly of the myristic acid conjugated peptide (MYR-5A) into nanomicellar structures and its characterization via steady-state and time-resolved fluorescence spectroscopy. The structural differences between the free peptide (5A) and MYR-5A conjugate were also probed, using tryptophan fluorescence, FÓ§rster resonance energy transfer (FRET), dynamic light scattering, and gel exclusion chromatography. To our knowledge, this is the first report of a lipoprotein assembly generated from a single ingredient and without a separate lipid component. The therapeutic utility of these nanoparticles (due to their capablity to incorporate a wide range of drugs into their core region for targeted delivery) was also investigated by probing the role of the scavenger receptor type B1 in this process. SIGNIFICANCE STATEMENT: Although lipoproteins have been considered as effective drug delivery agents, none of these nanoformulations has entered clinical trials to date. A major challenge to advancing lipoprotein-based formulations to the clinic has been the availability of a cost-effective protein or peptide constituent, needed for the assembly of the drug/lipoprotein nanocomplexes. This report of a robust, spontaneously assembling drug transport system from a single component could provide the template for a superior, targeted drug delivery strategy for therapeutics of cancer and other diseases (Counsell and Pohland, 1982).

New insight into the engineering of green carbon dots: Possible applications in emerging cancer theranostics.

Fluorescence imaging via carbon dots (CDs) has found multifarious applications in the biomedical sciences including biosensing, cancer cell bioimaging, drug delivery and tracking therapeutic response. Presently, the latest generation of fluorescence CDs known as green-CDs has attracted ever-increasing attention due to the use of natural sources, low-cost synthesis, nanoscale size, promising biocompatibility, superior photoluminescence, and ease of functionalization for versatile applications, which in turn could have higher priority over the traditional toxic fluorescent agents. In this review, we aim to have a new insight into the engineering green-CDs and their physicochemical properties. Moreover, we discuss the possible applications of green-CDs in self and active targeting, therapeutics delivery, and finally their promising future in cancer theranostics.

Facile Fabrication of Redox-Responsive Covalent Organic Framework Nanocarriers for Efficiently Loading and Delivering Doxorubicin.

Covalent organic frameworks (COFs) as drug delivery systems have shown great promise, but their pharmaceutical applications are often limited by complex building blocks, tedious preparations, irregular shape, and uncontrolled drug release within target cells. Herein, a facile strategy is developed to prepare PEGylated redox-responsive nanoscale COFs (denoted F68@SS-COFs) for efficiently loading and delivering doxorubicin (DOX) by use of FDA-approved Pluronic F68 and commercially available building blocks. The obtained F68@SS-COFs with controlled size, high stability, and good biocompatibility can not only achieve a very high DOX-loading content (about 21%) and very low premature leakage at physiological condition but can also rapidly respond to the tumor intracellular microenvironment and efficiently release DOX to kill tumor cells. Considering the readily available raw materials, simple preparation process, and desirable redox-responsiveness, the strategy provided here opens up a promising avenue to develop well-defined COFs-based nanomedicines for cancer therapy.

Preparation and Evaluation of Irinotecan Poly(Lactic-co-Glycolic Acid) Nanoparticles for Enhanced Anti-tumor Therapy.

Irinotecan (IRT), the pro-drug of SN-38, has exhibited potent cytotoxicity against various tumors. In order to enhance the anti-tumor effect of IRT, we prepared IRT-loaded PLGA nanoparticles (IRT-PLGA-NPs) by emulsion-solvent evaporation method. Firstly, IRT-PLGA-NPs were characterized through drug loading (DL), entrapment efficiency (EE), particle size, zeta potential, transmission electron microscopy (TEM), and differential scanning calorimetry (DSC). We next studied the in vitro release characteristics of IRT-PLGA-NPs. Finally, the pharmacokinetics and pharmacodynamics profiles of IRT-PLGA-NPs were investigated. The results revealed that IRT-PLGA-NPs were spherical with an average size of (169.97 ± 6.29) nm and its EE and DL were (52.22 ± 2.41)% and (4.75 ± 0.22)%, respectively. IRT-PLGA-NPs could continuously release drug for 14 days in vitro. In pharmacokinetics studies, for pro-drug IRT, the t1/2ß of IRT-PLGA-NPs was extended from 0.483 to 3.327 h compared with irinotecan solution (IRT-Sol), and for its active metabolite SN-38, the t1/2ß was extended from 1.889 to 4.811 h, which indicated that IRT-PLGA-NPs could prolong the retention times of both IRT and SN-38. The pharmacodynamics results revealed that the tumor doubling time, growth inhibition rate, and specific growth rate of IRT-PLGA-NPs were 2.13-, 1.30-, and 0.47-fold those of IRT-Sol, respectively, which demonstrated that IRT-PLGA-NPs could significantly inhibit the growth of tumor. In summary, IRT-PLGA-NPs, which exhibited excellent therapeutic effect against tumors, might be used as a potential carrier for tumor treatment in clinic.

Development of a novel conjugatable sunitinib analogue validated through in vitro and in vivo preclinical settings.

Sunitinib is an oral FDA/EMEA approved multi-targeted tyrosine kinase inhibitor. It possesses anti-angiogenic and antitumor activity against a variety of advanced solid tumors. However, its chemical core does not allow a potential linkage to tumor-homing elements that could eventually enhance its potency. Therefore, a novel linkable sunitinib derivative, designated SB1, was rationally designed and synthesized. The pharmaceutical profile of SB1 was explored both in vitro and in vivo. Mass spectrometry and NMR spectroscopy were utilized for characterization, while MTT assays and LC-MS/MS validated protocols were used to explore its antiproliferative effect and stability, respectively. Cytotoxicity evaluation in three glioma cells showed that SB1 preserved the antiproliferative effect of sunitinib. SB1 was stable in vitro after 24 h incubation in mouse plasma, while both agents exhibited bioequivalent pharmacokinetic characteristics after i.v. administration in Balb/c mice. To evaluate the levels of SB1 in mouse plasma, a novel analytical method was developed and validated in accordance to the US FDA and the EU EMA guidelines. We formulated a novel linkable sunitinib analog exhibiting similar antiproliferative and apoptotic properties with native sunitinib in glioma cell lines. Both SB1 and native sunitinib showed identical in vitro stability in mouse plasma and pharmacokinetics after i.v. administration in Balb/c mice.

Quantitative Assessment of Nanoparticle Biodistribution by Fluorescence Imaging, Revisited.

Fluorescence-based whole-body imaging is widely used in the evaluation of nanoparticles (NPs) in small animals, often combined with quantitative analysis to indicate their spatiotemporal distribution following systemic administration. An underlying assumption is that the fluorescence label represents NPs and the intensity increases with the amount of NPs and/or the labeling dyes accumulated in the region of interest. We prepare DiR-loaded poly(lactic- co-glycolic acid) (PLGA) NPs with different surface layers (polyethylene glycol with and without folate terminus) and compare the distribution of fluorescence signals in a mouse model of folate-receptor-expressing tumors by near-infrared fluorescence whole-body imaging. Unexpectedly, we observe that fluorescence distribution patterns differ far more dramatically with DiR loading than with the surface ligand, reaching opposite conclusions with the same type of NPs (tumor-specific delivery vs predominant liver accumulation). Analysis of DiR-loaded PLGA NPs reveals that fluorescence quenching, dequenching, and signal saturation, which occur with the increasing dye content and local NP concentration, are responsible for the conflicting interpretations. This study highlights the critical need for validating fluorescence labeling of NPs in the quantitative analysis of whole-body imaging. In light of our observation, we make suggestions for future whole-body fluorescence imaging in the in vivo evaluation of NP behaviors.

Poly(2-Ethyl-2-Oxazoline) as an Alternative to Poly(Vinylpyrrolidone) in Solid Dispersions for Solubility and Dissolution Rate Enhancement of Drugs.

Poly(2-ethyl-2-oxazoline) (PEOX), a biocompatible polymer considered as pseudopolypeptide, was introduced as a potential alternative to the commonly used polymer, poly(vinylpyrrolidone) (PVP) for the preparation of solid dispersion with a poorly soluble drug. Glipizide (GPZ), a Biopharmaceutical Classification System class II model drug, was selected for solubility and dissolution rate study. GPZ-polymer solid dispersions and physical mixtures were characterized and investigated by X-ray diffractometry, differential scanning calorimetry, scanning electron microscopy, and FTIR spectroscopy. The impact of polymers on crystal nucleation kinetics was studied, and PEOX exhibited strong inhibitory effect compared with PVP. Solubility and dissolution behavior of the prepared solid dispersions and their physical blends were in vitro examined and evaluated. A significant enhancement in GPZ solubility was obtained with PEOX compared with the pure drug and solid dispersion with PVP. A big improvement in the intrinsic dissolution rate (45 times) and dissolved amount of GPZ (58 times) was achieved with PEOX in fasted state simulated intestinal fluid, against comparable enhancement observed with PEOX and PVP in phosphate buffer at pH 6.8. Lower molecular weight of PEOX-5K (5000 g/mol) was found to be superior to higher molecular weight PEOX-50K (50,000 g/mol) in the improvement of dissolution behavior. The findings of this study with GPZ as a model drug introduce lower molecular weight PEOX as a promising polymeric carrier toward better oral bioavailability of poorly soluble drugs.

Preparation and in Vitro Analysis of Human Serum Albumin Nanoparticles Loaded with Anthracycline Derivatives.

Nanoparticles prepared using human serum albumin (HSA) have emerged as versatile carriers for improving the pharmacokinetic profile of drugs. The desolvation of HSA using ethanol followed by stabilization through crosslinking with glutaraldehyde is a common technique for preparing HSA nanoparticles, but our knowledge concerning the characteristics (or functions) of HSA nanoparticles and their efficiency when loaded with drugs is limited. To address this issue in more detail, we prepared anthracycline-loaded HSA nanoparticles. Doxorubicin-loaded HSA nanoparticles with a size similar to doxorubicin-unloaded particles could be prepared by desolvating at a higher pH (8-9), and the size (100-150 nm) was optimum for delivery to tumor tissues. Using this procedure, HSA nanoparticles were loaded with other anthracycline derivatives, and all showed cytotoxicity in cancer cells. However, the efficiency of drug loading and dissolution rate were different among them possibly due to the differences in the type of association of the drugs on nanoparticles (doxorubicin and daunorubicin; covalently bound to nanoparticles, pirarubicin; both covalently bound to and adsorbed on nanoparticles, aclarubicin; adsorbed on nanoparticles). Since the formulation of such drug-loaded HSA nanoparticles should be modified for efficient delivery to tumors, the findings reported herein provide the useful information for optimizing the formulation and the production process for the HSA nanoparticles using a desolvation technique.

Distribution of PLGA-modified nanoparticles in 3D cell culture models of hypo-vascularized tumor tissue.

BACKGROUND: Advanced stage cancer treatments are often invasive and painful-typically comprised of surgery, chemotherapy, and/or radiation treatment. Low transport efficiency during systemic chemotherapy may require high chemotherapeutic doses to effectively target cancerous tissue, resulting in systemic toxicity. Nanotherapeutic platforms have been proposed as an alternative to more safely and effectively deliver therapeutic agents directly to tumor sites. However, cellular internalization and tumor penetration are often diametrically opposed, with limited access to tumor regions distal from vasculature, due to irregular tissue morphologies. To address these transport challenges, nanoparticles (NPs) are often surface-modified with ligands to enhance transport and longevity after localized or systemic administration. Here, we evaluate stealth polyethylene-glycol (PEG), cell-penetrating (MPG), and CPP-stealth (MPG/PEG) poly(lactic-co-glycolic-acid) (PLGA) NP co-treatment strategies in 3D cell culture representing hypo-vascularized tissue. RESULTS: Smaller, more regularly-shaped avascular tissue was generated using the hanging drop (HD) method, while more irregularly-shaped masses were formed with the liquid overlay (LO) technique. To compare NP distribution differences within the same type of tissue as a function of different cancer types, we selected HeLa, cervical epithelial adenocarcinoma cells; CaSki, cervical epidermoid carcinoma cells; and SiHa, grade II cervical squamous cell carcinoma cells. In HD tumors, enhanced distribution relative to unmodified NPs was measured for MPG and PEG NPs in HeLa, and for all modified NPs in SiHa spheroids. In LO tumors, the greatest distribution was observed for MPG and MPG/PEG NPs in HeLa, and for PEG and MPG/PEG NPs in SiHa spheroids. CONCLUSIONS: Pre-clinical evaluation of PLGA-modified NP distribution into hypo-vascularized tumor tissue may benefit from considering tissue morphology in addition to cancer type.

Recent Advances of Individual BODIPY and BODIPY-Based Functional Materials in Medical Diagnostics and Treatment.

BACKGROUND: The group of fluorophores on boron dipyrrin platform (4,4- difluoro-4-bora3a,4a-diaza-s-indacene, also known as BODIPY) has attracted much attention in the field of molecular sensorics, including sensing of biomolecules and bioprocesses. Structural diversity of existing BODIPY with ample opportunities of directed modification of compounds makes this class of fluorophores attractive for medical and biological purposes. The recent progress in the design and functionalization of BODIPY allows using them for modification of drug micro- and nanocarriers in order to improve their therapeutic effect in cancer treatment. At the same time, integration of BODIPY into drug carriers provides the possibility of in vitro and in vivo real time imaging of used drug carriers. The high fluorescent intensity and low toxicity of BODIPY granted for conjugation with different biomolecules. RESULTS: The present review focuses on the recent advances for application of individual BODIPY in medical diagnostics, antimicrobial activity, as well as establishing the role of BODIPY in labeling of biomolecules (e.g. proteins, hormones and DNA). Also the review highlights the potential of BODIPY in functionalization of drug micro- and nanocarriers in order to achieve better therapeutic efficiency compared with non-modified materials. The advantages derived from the use of BODIPY for preparation and modification of drug carriers are critically evaluated and potential for future challenges, especially concerning the design of innovative multi-functional BODIPY-based nanocarriers, is discussed in detail using representative examples from literature. CONCLUSION: Our objective was to show that BODIPY are powerful tools for bioimaging, labeling of biomolecules and construction of new multifunctional drug carriers.

pH responsive controlled release of anti-cancer hydrophobic drugs from sodium alginate and hydroxyapatite bi-coated iron oxide nanoparticles.

Developing a drug carrier system which could perform targeted and controlled release over a period of time is utmost concern in the pharmaceutical industry. This is more relevant when designing drug carriers for poorly water soluble drug molecules such as curcumin and 6-gingerol. Development of a drug carrier system which could overcome these limitations and perform controlled and targeted drug delivery is beneficial. This study describes a promising approach for the design of novel pH sensitive sodium alginate, hydroxyapatite bilayer coated iron oxide nanoparticle composite (IONP/HAp-NaAlg) via the co-precipitation approach. This system consists of a magnetic core for targeting and a NaAlg/HAp coating on the surface to accommodate the drug molecules. The nanocomposite was characterized using FT-IR spectroscopy, X-ray diffraction, scanning electron microscopy, transmission electron microscopy and thermogravimetric analysis. The loading efficiency and loading capacity of curcumin and 6-gingerol were examined. In vitro drug releasing behavior of curcumin and 6-gingerol was studied at pH 7.4 and pH 5.3 over a period of seven days at 37°C. The mechanism of drug release from the nanocomposite of each situation was studied using kinetic models and the results implied that, the release is typically via diffusion and a higher release was observed at pH 5.3. This bilayer coated system can be recognized as a potential drug delivery system for the purpose of curcumin and 6-gingerol release in targeted and controlled manner to treat diseases such as cancer.

Label-free visualization of nilotinib-functionalized gold nanoparticles within single mammalian cells by C60- SIMS imaging.

Obtaining a comprehensive grasp of the behavior and interaction of pharmaceutical compounds within single cells provides some of the fundamental details necessary for more effective drug development. In particular, the changes ensuing in the carrier, drug, and host environment in targeted drug therapy applications must be explored in greater detail, as these are still not well understood. Here, nilotinib-functionalized gold nanoparticles are examined within single mammalian cells with use of imaging cluster secondary ion mass spectrometry in a model study designed to enhance our understanding of what occurs to these particles once that have been internalized. Nilotinib, several types of gold nanoparticles, and the functionalized combination of the two were surveyed and successfully imaged within single cells to determine uptake and performance. Both nilotinib and the gold particle are able to be distinguished and visualized in the functionalized nanoparticle assembly within the cell. These compounds, while both internalized, do not appear to be present in the same pixels of the chemical image, indicating possible cleavage of nilotinib from the particle after cell uptake. The method provided in this work is a direct measurement of uptake and subcellular distribution of an active drug and its carrier within a framework. The results obtained from this study have the potential to be applied to future studies to provide more effective and specific cellular delivery of a relevant pharmaceutical compound.