Engineered 3D Silk‐Based Metastasis Models: Interactions Between Human Breast Adenocarcinoma,Mesenchymal Stem Cells and Osteoblast‐Like Cells

Bone metastasis occurs in 70% of breast cancer patients and is a frequent cause of morbidity in cancer patients. A delicate balance exists in the bone microenvironment, but the functional dynamics underlying the tumor cell‐microenvironment interactions remain poorly understood. 3D in vitro model systems of metastasis can throw new light on this phenomenon. Silk protein fibroin scaffolds, are cytocompatible for 3D cancer cell culture. They are structurally more resistant to protease degradation than other native biomaterials making these matrices suitable for cancer modeling. In this report, human breast adenocarcinoma cells, human osteoblast like cells and mesenchymal stem cells are co‐cultered. Cancer cells and osteoblast‐like cells are found to interact through secreted products. Decreased population of osteoblast‐like cells and mineralization of extracellular matrix are observed as a result of co‐culture. Significantly increased migration of breast cancer cells is observed in the bone‐like constructs than in non‐seeded scaffolds. The co‐culture constructs show significant increase in drug resistance, invasiveness and angiogenicity. Co‐culture of breast cancer cells with osteoblast like cells and mesenchymal stem cells also indicate that the interaction of cancer cells with bone microenvironment varies with spatial organization, presence of osteogenic factors as well as stromal cell type. Here, results show that 3D in vitro co‐culture models is possibly a better system to study and target cancer progression.     

The Application of Stimuli‐Responsive VEGF‐ and ATP‐Aptamer‐Based Microcapsules for the Controlled Release of an Anticancer Drug,and the Selective Targeted Cytotoxicity toward Cancer Cells

The synthesis of microcapsules consisting of DNA shells crosslinked by anti‐VEGF (vascular epithelial growth factor) or anti‐ATP (adenosine triphosphate) aptamers and loaded with tetramethylrhodamine‐modified dextran, TMR‐D, and Texas Red‐modified dextran, TR‐D, respectively, as fluorescence labels acting as models for drug loads, is described. The aptamer‐functionalized microcapsules act as stimuli‐responsive carriers for the triggered release of the fluorescent labels in the presence of the overexpressed cancer cell biomarkers VEGF or ATP. The VEGF‐ and ATP‐responsive microcapsules are, also, loaded with the anticancer drug doxorubicin (DOX), in the form of DOX‐functionalized dextran, DOX‐D. The release of DOX‐D from the respective microcapsules proceeds in the presence of VEGF or ATP as triggers. Preliminary cell experiments reveal that the ATP‐responsive DOX‐D‐loaded microcapsules undergo effective endocytosis into MDA‐MB‐231 cancer cells. The ATP‐responsive DOX‐D‐loaded microcapsules incorporated into the MDA‐MB‐231 cancer cells reveal impressive cytotoxicity as compared to normal epithelial MCF‐10A breast cells (50% vs 0% cell death after 24 h, respectively). The cytotoxicity of the ATP‐responsive DOX‐D‐loaded microcapsules toward the cancer cells is attributed to the effective unlocking of the microcapsules by overexpressed ATP, and to the subsequent release of DOX from the dextran backbone under acidic conditions present in cancer cells (pH = 6.2).     

Inhibition of Cancer Cell Migration by Gold Nanorods: Molecular Mechanisms and Implications for Cancer Therapy

Gold nanorods have received much attention because of their distinct physicochemical properties and promising applications in bioimaging, biosensing, drug delivery, photothermal therapy, and optoelectronic devices. However, little is known regarding their effect on tumor metastasis. In the present investigation, serum protein‐coated gold nanorods (AuNRs) at low concentrations is shown to exhibit no apparent effects on the viability and proliferation of three different metastatic cancer cell lines, that is, MDA‐MB‐231 human breast cancer cells, PC3 human prostate cancer cells, and B16F10 mouse melanoma cells, but effectively inhibit their migration and invasion in vitro. Quantitative proteomics and real‐time PCR array analyses indicate that exposure of cells to AuNRs can down‐regulate the expression of diverse energy generation‐related genes, which accounts for their inhibition of mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis. The impairment of OXPHOS and glycolysis results in a distinctive reduction of ATP production and subsequent inhibition of F‐actin cytoskeletal assembly, which is crucial for the migration and invasion of cancer cells. The inhibitory effect of AuNRs on cancer cell migration is also confirmed in vivo. Taken together, the unique mechanism in inhibiting cancer cell migration by AuNRs might provide a new approach to specific cancer therapeutic treatment.     

Tuning the Microenvironment: Click‐Crosslinked Hyaluronic Acid‐Based Hydrogels Provide a Platform for Studying Breast Cancer Cell Invasion

A big challenge in cell culture is the non‐natural environment in which cells are routinely screened, making in vivo phenomena, such as cell invasion, difficult to understand and predict. To study cancer cell invasion, extracellular matrix (ECM) analogs with decoupled mechanical and chemical properties are required. Hyaluronic acid (HA)‐based hydrogels crosslinked with matrix‐metalloproteinase (MMP)‐cleavable peptides are developed to study MDA‐MB‐231 breast cancer cell invasion. Hydrogels are synthesized by reacting furan‐modified HA with bismaleimide peptide crosslinkers in a Diels–Alder click reaction. This new hydrogel takes advantage of the biomimetic properties of HA, which is overexpressed in breast cancer, and eliminates the use of nonadhesive crosslinkers, such as poly(ethylene glycol) (PEG). The crosslink (mechanical) and ligand (chemical) densities are varied independently to evaluate the effects of each parameter on cell migration. Increased crosslink density correlates with decreased MDA‐MB‐231 cell invasion whereas incorporation of MMP‐cleavable sequences within the peptide crosslinker enhances invasion. Increasing the ligand density of pendant GRGDS groups induces cell proliferation, but has no significant impact on invasion. By independently tuning the mechanical and chemical environment of ECM mimetic hydrogels, a platform is provided that recapitulates variable tissue properties and elucidates the role of the microenvironment in cancer cell invasion.     

Enzyme‐Driven Release of Loads from Nucleic Acid–Capped Metal–Organic Framework Nanoparticles

Nucleic acid–modified UiO‐68 metal–organic framework nanoparticles, NMOFs, are loaded with the anticancer drug camptothecin (or drug models), and the loaded NMOFs are capped with sequence‐specific duplex units. The NMOFs are unlocked by the biocatalytic decomposition of the duplex capping units that result in the release of the drug (or drug models). The enzymes used are DNase I, a nicking enzyme (Nt.BbvCI), an endonuclease (EcoRI), and an exonuclease III (Exo III). Camptothecin‐loaded NMOFs, capped by tailored hairpin nucleic acids being cooperatively unlocked by adenosine triphosphate (ATP), that is overexpressed in cancer cells, and Exo III are prepared. The camptothecin‐loaded NMOFs reveal that selective cytotoxicity toward MDA‐MB‐231 cancer cells and ≈55% apoptosis of the cancer cells is observed after 5 days of treatment with the NMOFs, while only ≈15% apoptosis of epithelial MCF‐10A breast cells is observed.     

ATP‐Responsive Aptamer‐Based Metal–Organic Framework Nanoparticles (NMOFs) for the Controlled Release of Loads and Drugs

Nanoparticles consisting of metal–organic frameworks (NMOFs) modified with nucleic acid binding strands are synthesized. The NMOFs are loaded with a fluorescent agent or with the anticancer drug doxorubicin, and the loaded NMOFs are capped by hybridization with a complementary nucleic acid that includes the ATP‐aptamer or the ATP‐AS1411 hybrid aptamer in caged configurations. The NMOFs are unlocked in the presence of ATP via the formation of ATP‐aptamer complexes, resulting in the release of the loads. As ATP is overexpressed in cancer cells, and since the AS1411 aptamer recognizes the nucleolin receptor sites on the cancer cell membrane, the doxorubicin‐loaded NMOFs provide functional carriers for targeting and treatment of cancer cells. Preliminary cell experiments reveal impressive selective permeation of the NMOFs into MDA‐MB‐231 breast cancer cells as compared to MCF‐10A normal epithelial breast cells. High cytotoxic efficacy and targeted drug release are observed with the ATP‐AS1411‐functionalized doxorubicin‐loaded NMOFs.     

Gel–Liposome‐Mediated Co‐Delivery of Anticancer Membrane‐Associated Proteins and Small‐Molecule Drugs for Enhanced Therapeutic Efficacy

A programmed drug‐delivery system that can transport different anticancer therapeutics to their distinct targets holds vast promise for cancer treatment. Herein, a core–shell‐based “nanodepot” consisting of a liposomal core and a crosslinked‐gel shell (designated Gelipo) is developed for the sequential and site‐specific delivery (SSSD) of tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) and doxorubicin (Dox). As a small‐molecule drug intercalating the nuclear DNA, Dox is loaded in the aqueous core of the liposome, while TRAIL, acting on the death receptor (DR) on the plasma membrane, is encapsulated in the outer shell made of crosslinked hyaluronic acid (HA). The degradation of the HA shell by HAase that is concentrated in the tumor environment results in the rapid extracellular release of TRAIL and subsequent internalization of the liposomes. The parallel activity of TRAIL and Dox show synergistic anticancer efficacy. The half‐maximal inhibitory concentration (IC₅₀) of TRAIL and Dox co‐loaded Gelipo (TRAIL/Dox‐Gelipo) toward human breast cancer (MDA‐MB‐231) cells is 83 ng mL^–1 (Dox concentration), which presents a 5.9‐fold increase in the cytotoxicity compared to 569 ng mL^–1 of Dox‐loaded Gelipo (Dox‐Gelipo). Moreover, with the programmed choreography, Gelipo significantly improves the inhibition of the tumor growth in the MDA‐MB‐231 xenograft tumor animal model.     

Direct‐Write Assembly of Microperiodic Silk Fibroin Scaffolds for Tissue Engineering Applications

Three–dimensional, microperiodic scaffolds of regenerated silk fibroin have been fabricated for tissue engineering by direct ink writing. The ink, which consisted of silk fibroin solution from the Bombyx mori silkworm, was deposited in a layer‐by‐layer fashion through a fine nozzle to produce a 3D array of silk fibers of diameter 5 µm. The extruded fibers crystallized when deposited into a methanol‐rich reservoir, retaining a pore structure necessary for media transport. The rheological properties of the silk fibroin solutions were investigated and the crystallized silk fibers were characterized for structure and mechanical properties by infrared spectroscopy and nanoindentation, respectively. The scaffolds supported human bone marrow‐derived mesenchymal stem cell (hMSC) adhesion, and growth. Cells cultured under chondrogenic conditions on these scaffolds supported enhanced chondrogenic differentiation based on increased glucosaminoglycan production compared to standard pellet culture. Our results suggest that 3D silk fibroin scaffolds may find potential application as tissue engineering constructs due to the precise control of their scaffold architecture and their biocompatibility.     

Biocompatible,Uniform, and Redispersible Mesoporous Silica Nanoparticles for Cancer‐Targeted Drug Delivery In Vivo

Engineering multifunctional nanocarriers for targeted drug delivery shows promising potentials to revolutionize the cancer chemotherapy. Simple methods to optimize physicochemical characteristics and surface composition of the drug nanocarriers need to be developed in order to tackle major challenges for smooth translation of suitable nanocarriers to clinical applications. Here, rational development and utilization of multifunctional mesoporous silica nanoparticles (MSNPs) for targeting MDA‐MB‐231 xenograft model breast cancer in vivo are reported. Uniform and redispersible poly(ethylene glycol)‐incorporated MSNPs with three different sizes (48, 72, 100 nm) are synthesized. They are then functionalized with amino‐β‐cyclodextrin bridged by cleavable disulfide bonds, where amino‐β‐cyclodextrin blocks drugs inside the mesopores. The incorporation of active folate targeting ligand onto 48 nm of multifunctional MSNPs (PEG‐MSNPs48‐CD‐PEG‐FA) leads to improved and selective uptake of the nanoparticles into tumor. Targeted drug delivery capability of PEG‐MSNPs48‐CD‐PEG‐FA is demonstrated by significant inhibition of the tumor growth in mice treated with doxorubicin‐loaded nanoparticles, where doxorubicin is released triggered by intracellular acidic pH and glutathione. Doxorubicin‐loaded PEG‐MSNPs48‐CD‐PEG‐FA exhibits better in vivo therapeutic efficacy as compared with free doxorubicin and non‐targeted nanoparticles. Current study presents successful utilization of multifunctional MSNP‐based drug nanocarriers for targeted cancer therapy in vivo.     

Microphysiological Systems as Enabling Tools for Modeling Complexity in the Tumor Microenvironment and Accelerating Cancer Drug Development

Tumor cell heterogeneity with distinct phenotypes, genotypes, and epigenetic states as well as the complex tumor microenvironment is major challenges for cancer diagnosis and treatment. There have been substantial advances in our knowledge of tumor biology and in the capabilities of available biological analysis tools; however, the absence of physiologically relevant in vitro testing platforms limits our ability to gain an in‐depth understanding of the role of the tumor microenvironment in cancer pathology. In this review, recent advances in engineered tumor microenvironments to advance cancer research and drug discovery are presented, including tumor spheroids, microfluidic chips, paper scaffolds, hydrogel‐based engineered tissues, 3D bioprinted scaffolds, and multiscale topography. Furthermore, how these technologies address the specific characteristics of the native tumor microenvironment is described. Through the comparison of these biomimetic 3D tumor models to conventional 2D culture models, the validity and physiological relevance of these platforms for fundamental in vitro studies of the tumor biology, as well as their potential use in drug screening applications, is also discussed.     

Self‐Assembling Doxorubicin Silk Hydrogels for the Focal Treatment of Primary Breast Cancer

Standard care for early stage breast cancer includes tumor resection and local radiotherapy to achieve long‐term remission. Systemic chemotherapy provides only low locoregional control of the disease; to address this, self‐assembling silk hydrogels that can retain and then deliver doxorubicin locally are described. Self‐assembling silk hydrogels show no swelling, are readily loaded with doxorubicin under aqueous conditions, and release drug over 4 weeks in amounts that can be fine‐tuned by varying the silk content. Following successful in vitro studies, locally injected silk hydrogels loaded with doxorubicin show excellent antitumor response in mice, outperforming the equivalent amount of doxorubicin delivered intravenously. In addition to reducing primary tumor growth, doxorubicin‐loaded silk hydrogels reduce metastatic spread and are well tolerated in vivo. Thus, silk hydrogels are well suited for the local delivery of chemotherapy and provide a promising approach to improve locoregional control of breast cancer.     

Stimuli‐Responsive Nucleic Acid‐Based Polyacrylamide Hydrogel‐Coated Metal–Organic Framework Nanoparticles for Controlled Drug Release

The synthesis of doxorubicin‐loaded metal–organic framework nanoparticles (NMOFs) coated with a stimuli‐responsive nucleic acid‐based polyacrylamide hydrogel is described. The formation of the hydrogel is stimulated by the crosslinking of two polyacrylamide chains, P_A and P_B, that are functionalized with two nucleic acid hairpins ( 4 ) and ( 5 ) using the strand‐induced hybridization chain reaction. The resulting duplex‐bridged polyacrylamide hydrogel includes the anti‐ATP (adenosine triphosphate) aptamer sequence in a caged configuration. The drug encapsulated in the NMOFs is locked by the hydrogel coating. In the presence of ATP that is overexpressed in cancer cells, the hydrogel coating is degraded via the formation of the ATP–aptamer complex, resulting in the release of doxorubicin drug. In addition to the introduction of a general means to synthesize drug‐loaded stimuli‐responsive nucleic acid‐based polyacrylamide hydrogel‐coated NMOFs hybrids, the functionalized NMOFs resolve significant limitations associated with the recently reported nucleic acid‐gated drug‐loaded NMOFs. The study reveals substantially higher loading of the drug in the hydrogel‐coated NMOFs as compared to the nucleic acid‐gated NMOFs and overcomes the nonspecific leakage of the drug observed with the nucleic‐acid‐protected NMOFs. The doxorubicin‐loaded, ATP‐responsive, hydrogel‐coated NMOFs reveal selective and effective cytotoxicity toward MDA‐MB‐231 breast cancer cells, as compared to normal MCF‐10A epithelial breast cells.     

Nanometric Micelles with Photo‐Triggered Cytotoxicity

The development of a photo‐responsive micellar system capable of triggering cell death is reported. Precursors of the micelles are synthesized by connecting a lipophilic chain to a hydrophilic polyethylene glycol via a photo‐labile nitrobenzyl group. The resulting amphiphilic units are self‐assembled in water forming 12 nm micelles that are readily internalized into cells. Upon photo‐irradiation, micelles undergo cleavage and yield a cytotoxic nitrosobenzaldehyde derivative, which significantly inhibits the proliferation of MDA‐MB‐231 cells under standard in vitro conditions.     

High Throughput,Polymeric Aqueous Two‐Phase Printing of Tumor Spheroids

This paper presents a new 3D culture microtechnology for high throughput production of tumor spheroids and validates its utility for screening anti‐cancer drugs. Two immiscible polymeric aqueous solutions are used and a submicroliter drop of the “patterning” phase containing cells is microprinted into a bath of the “immersion” phase. Selecting proper formulations of biphasic systems using a panel of biocompatible polymers results in the formation of a round drop that confines cells to facilitate spontaneous formation of a spheroid without any external stimuli. Adapting this approach to robotic tools enables straightforward generation and maintenance of spheroids of well‐defined size in standard microwell plates and biochemical analysis of spheroids in situ, which is not possible with existing techniques for spheroid culture. To enable high throughput screening, a phase diagram is established to identify minimum cell densities within specific volumes of the patterning drop to result in a single spheroid. Spheroids show normal growth over long‐term incubation and dose‐dependent decrease in cellular viability when treated with drug compounds, but present significant resistance compared to monolayer cultures. The unprecedented ease of implementing this microtechnology and its robust performance will benefit high throughput studies of drug screening against cancer cells with physiologically relevant 3D tumor models.     

Cell‐Tethered Ligands Modulate Bone Remodeling by Osteoblasts and Osteoclasts

The goals of the present study are to establish an in vitro co‐culture model of osteoblast and osteoclast function and to quantify the resulting bone remodeling. The bone is tissue engineered using well‐defined silk protein biomaterials in 2D and 3D formats in combination with human cells. Parathyroid hormone (PTH) and glucose‐dependent insulinotropic peptide (GIP) are selected because of their roles in bone remodeling for expression in tethered format on human mesenchymal stem cells (hMSCs). The cell‐modified biomaterial surfaces are reconstructed from scanning electron microscopy images into 3D models for quantitative measurement of surface characteristics. Increased calcium deposition and surface roughness are found in 3D surface models of silk protein films remodeled by co‐cultures containing tethered PTH, and decreased surface roughness is found for the films remodeled by tethered GIP co‐cultures. Increased surface roughness is not found in monocultures of hMSCs expressing tethered PTH, suggesting that osteoclast‐osteoblast interactions in the presence of PTH signaling are responsible for the increased mineralization. These data point towards the design of in vitro bone models in which osteoblast‐osteoclast interactions are mimicked for a better understanding of bone remodeling.     

Cancer Cell Membrane‐Coated Gold Nanocages with Hyperthermia‐Triggered Drug Release and Homotypic Target Inhibit Growth and Metastasis of Breast Cancer

The cell‐specific targeting drug delivery and controlled release of drug at the cancer cells are still the main challenges for anti‐breast cancer metastasis therapy. Herein, the authors first report a biomimetic drug delivery system composed of doxorubicin (DOX)‐loaded gold nanocages (AuNs) as the inner cores and 4T1 cancer cell membranes (CMVs) as the outer shells (coated surface of DOX‐incorporated AuNs (CDAuNs)). The CDAuNs, perfectly utilizing the natural cancer cell membranes with the homotypic targeting and hyperthermia‐responsive ability to cap the DAuNs with the photothermal property, can realize the selective targeting of the homotypic tumor cells, hyperthermia‐triggered drug release under the near‐infrared laser irradiation, and the combination of chemo/photothermal therapy. The CDAuNs exhibit a stimuli‐release of DOX under the hyperthermia and a high cell‐specific targeting of the 4T1 cells in vitro. Moreover, the excellent combinational therapy with about 98.9% and 98.5% inhibiting rates of the tumor volume and metastatic nodules is observed in the 4T1 orthotopic mammary tumor models. As a result, CDAuNs can be a promising nanodelivery system for the future therapy of breast cancer.     

Fabrication of Silk Microneedles for Controlled‐Release Drug Delivery

Microneedles are emerging as a minimally invasive drug delivery alternative to hypodermic needles. Current material systems utilized in microneedles impose constraints hindering the further development of this technology. In particular, it is difficult to preserve sensitive biochemical compounds (such as pharmaceuticals) during processing in a single microneedle system and subsequently achieve their controlled release. A possible solution involves fabricating microneedles systems from the biomaterial silk fibroin. Silk fibroin combines excellent mechanical properties, biocompatibility, biodegradability, benign processing conditions, and the ability to preserve and maintain the activity of biological compounds entrained in its material matrix. The degradation rate of silk fibroin and the diffusion rate of the entrained molecules can be controlled simply by adjusting post‐processing conditions. This combination of properties makes silk an ideal choice to improve on existing issues associated with other microneedle‐based drug delivery system. In this study, a fabrication method to produce silk biopolymer microstructures with the high aspect ratios and mechanical properties required to manufacture microneedle systems is reported. Room temperature and aqueous‐based micromolding allows for the bulk loading of these microneedles with labile drugs. The drug release rate is decreased 5.6‐fold by adjusting the post‐processing conditions of the microneedles, mainly by controlling the silk protein secondary structure. The release kinetics are quantified in an in vitro collagen hydrogel model, which allows tracking of the model drug. Antibiotic loaded silk microneedles are manufactured and used to demonstrate a 10‐fold reduction of bacterial density after their application. The processing strategies developed in this study can be expanded to other silk‐based structural formats for drug delivery and biologicals storage applications.     

MUC1 Aptamer Targeted SERS Nanoprobes

Recently, surface‐enhanced Raman scattering (SERS) nanoprobes (NPs) have shown promise in the field of cancer imaging due to their unparalleled signal specificity and high sensitivity. This study reports the development of a DNA aptamer targeted SERS NP. Recently, aptamers are being investigated as a viable alternative to more traditional antibody targeting due to their low immunogenicity and low cost of production. A strategy is developed to functionalize SERS NPs with DNA aptamers, which target Mucin1 (MUC1) in human breast cancer (BC). Thorough in vitro characterization studies demonstrate excellent serum stability and specific binding of the targeted NPs to MUC1. In order to test their in vivo targeting capability, MUC1‐targeted SERS NPs are coinjected with nontargeted or blocked MUC1‐targeted SERS NPs in BC xenograft mouse models. A two‐tumor mouse model with differential expression of MUC1 (MDA‐MB‐468 and MDA‐MB‐453) is used to control for active versus passive targeting in the same animals. The results show that the targeted SERS NPs home to the tumors via active targeting of MUC1, with low levels of passive targeting. This strategy is expected to be an advantageous alternative to antibody‐based targeting and useful for targeted imaging of tumor extent, progression, and therapeutic response.     

Nonmulberry Silk Based Ink for Fabricating Mechanically Robust Cardiac Patches and Endothelialized Myocardium‐on‐a‐Chip Application

Bioprinting holds great promise toward engineering functional cardiac tissue constructs for regenerative medicine and as drug test models. However, it is highly limited by the choice of inks that require maintaining a balance between the structure and functional properties associated with the cardiac tissue. In this regard, a novel and mechanically robust biomaterial‐ink based on nonmulberry silk fibroin protein is developed. The silk‐based ink demonstrates suitable mechanical properties required in terms of elasticity and stiffness (≈40 kPa) for developing clinically relevant cardiac tissue constructs. The ink allows the fabrication of stable anisotropic scaffolds using a dual crosslinking method, which are able to support formation of aligned sarcomeres, high expression of gap junction proteins as connexin‐43, and maintain synchronously beating of cardiomyocytes. The printed constructs are found to be nonimmunogenic in vitro and in vivo. Furthermore, delving into an innovative method for fabricating a vascularized myocardial tissue‐on‐a‐chip, the silk‐based ink is used as supporting hydrogel for encapsulating human induced pluripotent stem cell derived cardiac spheroids (hiPSC‐CSs) and creating perfusable vascularized channels via an embedded bioprinting technique. The ability is confirmed of silk‐based supporting hydrogel toward maturation and viability of hiPSC‐CSs and endothelial cells, and for applications in evaluating drug toxicity.     

Polymer Nanoparticles Encased in a Cyclodextrin Complex Shell for Potential Site‐ and Sequence‐Specific Drug Release

Time‐staggered combination chemotherapy strategies show immense potential in cell culture systems, but fail to successfully translate clinically due to different routes of administration and disparate formulation parameters that preclude a specific order of drug presentation. A novel platform consisting of drug‐containing PLGA polymer nanoparticles, stably fashioned with a shell composed of drug complexed with cationic cyclodextrin, capable of releasing drugs time‐ and sequence‐specifically within tumors is designed. Morphological examination of nanoparticles measuring 150 nm highlight stable and distinct compartmentalization of model drugs, rhodamine and bodipy, within the core and shell, respectively. Sequential release is observed in vitro, owing to cyclodextrin shell displacement and subsequent sustained release of core‐loaded drug, kinetics preserved in breast cancer cells following internalization. Importantly, time‐staggered release is corroborated in a murine breast cancer model following intravenous administration. Precise control of drug release order, site‐specifically, potentially opens novel avenues in polychemotherapy for synergy and chemosensitization strategies.