The T-cell niche tunes immune function through modulation of the cytoskeleton and TCR-antigen forces.

Obesity is a major public health crisis given its rampant growth and association with an increased risk for cancer. Interestingly, patients with obesity tend to have an increased tumor burden and decreased T-cell function. It remains unclear how obesity compromises T-cell mediated immunity. To address this question, we modeled the adipocyte niche using the secretome released from adipocytes as well as the niche of stromal cells and investigated how these factors modulated T-cell function. We found that the secretomes altered antigen-specific T-cell receptor (TCR) triggering and activation. RNA-sequencing analysis identified thousands of gene targets modulated by the secretome including those associated with cytoskeletal regulation and actin polymerization. We next used molecular force probes to show that T-cells exposed to the adipocyte niche display dampened force transmission to the TCR-antigen complex and conversely, stromal cell secreted factors lead to significantly enhanced TCR forces. These results were then validated in diet-induced obese mice. Importantly, secretome-mediated TCR force modulation mirrored the changes in T-cell functional responses in human T-cells using the FDA-approved immunotherapy, blinatumomab. Thus, this work shows that the adipocyte niche contributes to T-cell dysfunction through cytoskeletal modulation and reduces TCR triggering by dampening TCR forces consistent with the mechanosensor model of T-cell activation.

Engineering Improved CAR T Cell Products with A Multi-Cytokine Particle Platform for Hematologic and Solid Tumors.

Despite the remarkable clinical efficacy of chimeric antigen receptor (CAR) T cells in hematological malignancies, only a subset of patients achieves a durable complete response (dCR). DCR has been correlated with CAR T cell products enriched with T cells memory phenotypes. Therefore, reagents that consistently promote memory phenotypes during the manufacturing of CAR T cells have the potential to significantly improve clinical outcomes. A novel modular multi-cytokine particle (MCP) platform is developed that combines the signals necessary for activation, costimulation, and cytokine support into a single "all-in-one" stimulation reagent for CAR T cell manufacturing. This platform allows for the assembly and screening of compositionally diverse MCP libraries to identify formulations tailored to promote specific phenotypes with a high degree of flexibility. The approach is leveraged to identify unique MCP formulations that manufacture CAR T cell products from diffuse large B cell patients   with increased proportions of memory-like phenotypes MCP-manufactured CAR T cells demonstrate superior anti-tumor efficacy in mouse models of lymphoma and ovarian cancer through enhanced persistence. These findings serve as a proof-of-principle of the powerful utility of the MCP platform to identify "all-in-one" stimulation reagents that can improve the effectiveness of cell therapy products through optimal manufacturing.

Constitutively Synergistic Multiagent Drug Formulations Targeting MERTK, FLT3, and BCL-2 for Treatment of AML.

PURPOSE: Although high-dose, multiagent chemotherapy has improved leukemia survival rates, treatment outcomes remain poor in high-risk subsets, including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) in infants. The development of new, more effective therapies for these patients is therefore an urgent, unmet clinical need. METHODS: The dual MERTK/FLT3 inhibitor MRX-2843 and BCL-2 family protein inhibitors were screened in high-throughput against a panel of AML and MLL-rearranged precursor B-cell ALL (infant ALL) cell lines. A neural network model was built to correlate ratiometric drug synergy and target gene expression. Drugs were loaded into liposomal nanocarriers to assess primary AML cell responses. RESULTS: MRX-2843 synergized with venetoclax to reduce AML cell density in vitro. A neural network classifier based on drug exposure and target gene expression predicted drug synergy and growth inhibition in AML with high accuracy. Combination monovalent liposomal drug formulations delivered defined drug ratios intracellularly and recapitulated synergistic drug activity. The magnitude and frequency of synergistic responses were both maintained and improved following drug formulation in a genotypically diverse set of primary AML bone marrow specimens. CONCLUSIONS: We developed a nanoscale combination drug formulation that exploits ectopic expression of MERTK tyrosine kinase and dependency on BCL-2 family proteins for leukemia cell survival in pediatric AML and infant ALL cells. We demonstrate ratiometric drug delivery and synergistic cell killing in AML, a result achieved by a systematic, generalizable approach of combination drug screening and nanoscale formulation that may be extended to other drug pairs or diseases in the future.

Development of constitutively synergistic nanoformulations to enhance chemosensitivity in T-cell leukemia.

Advances in multiagent chemotherapy have led to recent improvements in survival for patients with acute lymphoblastic leukemia (ALL); however, a significant fraction do not respond to frontline chemotherapy or later relapse with recurrent disease, after which long-term survival rates remain low. To develop new, effective treatment options for these patients, we conducted a series of high-throughput combination drug screens to identify chemotherapies that synergize in a lineage-specific manner with MRX-2843, a small molecule dual MERTK and FLT3 kinase inhibitor currently in clinical testing for treatment of relapsed/refractory leukemias and solid tumors. Using experimental and computational approaches, we found that MRX-2843 synergized strongly-and in a ratio-dependent manner-with vincristine to inhibit both B-ALL and T-ALL cell line expansion. Based on these findings, we developed multiagent lipid nanoparticle formulations of these drugs that not only delivered defined drug ratios intracellularly in T-ALL, but also improved anti-leukemia activity following drug encapsulation. Synergistic and additive interactions were recapitulated in primary T-ALL patient samples treated with MRX-2843 and vincristine nanoparticle formulations, suggesting their clinical relevance. Moreover, the nanoparticle formulations reduced disease burden and prolonged survival in an orthotopic murine xenograft model of early thymic precursor T-ALL (ETP-ALL), with both agents contributing to therapeutic activity in a dose-dependent manner. In contrast, nanoparticles containing MRX-2843 alone were ineffective in this model. Thus, MRX-2843 increased the sensitivity of ETP-ALL cells to vincristine in vivo. In this context, the additive particles, containing a higher dose of MRX-2843, provided more effective disease control than the synergistic particles. In contrast, particles containing an even higher, antagonistic ratio of MRX-2843 and vincristine were less effective. Thus, both the drug dose and the ratio-dependent interaction between MRX-2843 and vincristine significantly impacted therapeutic activity in vivo. Together, these findings present a systematic approach to high-throughput combination drug screening and multiagent drug delivery that maximizes the therapeutic potential of combined MRX-2843 and vincristine in T-ALL and describe a novel translational agent that could be used to enhance therapeutic responses to vincristine in patients with T-ALL. This broadly generalizable approach could also be applied to develop other constitutively synergistic combination products for the treatment of cancer and other diseases.

Constitutively synergistic multiagent drug formulations targeting MERTK, FLT3, and BCL-2 for treatment of AML.

Although high-dose, multi-agent chemotherapy has improved leukemia survival rates in recent years, treatment outcomes remain poor in high-risk subsets, including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) in infants. Development of new, more effective therapies for these patients is therefore an urgent, unmet clinical need. To address this challenge, we developed a nanoscale combination drug formulation that exploits ectopic expression of MERTK tyrosine kinase and dependency on BCL-2 family proteins for leukemia cell survival in pediatric AML and MLL- rearranged precursor B-cell ALL (infant ALL). In a novel, high-throughput combination drug screen, the MERTK/FLT3 inhibitor MRX-2843 synergized with venetoclax and other BCL-2 family protein inhibitors to reduce AML cell density in vitro . Neural network models based on drug exposure and target gene expression were used to identify a classifier predictive of drug synergy in AML. To maximize the therapeutic potential of these findings, we developed a combination monovalent liposomal drug formulation that maintains ratiometric drug synergy in cell-free assays and following intracellular delivery. The translational potential of these nanoscale drug formulations was confirmed in a genotypically diverse set of primary AML patient samples and both the magnitude and frequency of synergistic responses were not only maintained but were improved following drug formulation. Together, these findings demonstrate a systematic, generalizable approach to combination drug screening, formulation, and development that maximizes therapeutic potential, was effectively applied to develop a novel nanoscale combination therapy for treatment of AML, and could be extended to other drug combinations or diseases in the future.

Multicolor Light-Induced Immune Activation via Polymer Photocaged Cytokines.

Cytokines act as potent, extracellular signals of the human immune system and can elicit striking treatment responses in patients with autoimmune disease, tissue damage, and cancer. Yet, despite their therapeutic potential, recombinant cytokine-mediated immune responses remain difficult to control as their administration is often systemic, whereas their intended sites of action are localized. To address the challenge of spatially and temporally constraining cytokine signals, we recently devised a strategy whereby recombinant cytokines are reversibly inactivated via chemical modification with photo-labile polymers that respond to visible LED light. Extending this approach to enable both in vivo and multicolor immune activation, here we describe a strategy whereby cytokines appended with heptamethine cyanine-polyethylene glycol are selectively re-activated ex vivo using tissue-penetrating near-infrared (NIR) light. We show that NIR LED light illumination of caged, pro-inflammatory cytokines restores cognate receptor signaling and potentiates the activity of T cell-engager cancer immunotherapies ex vivo. Using combinations of visible- and NIR-responsive cytokines, we further demonstrate multiwavelength optical control of T cell cytolysis ex vivo, as well as the ability to perform Boolean logic using multicolored light and orthogonally photocaged cytokine pairs as inputs and T cell activity as outputs. Together, this work demonstrates a novel approach to control extracellular immune cell signals using light, a strategy that in the future may improve our understanding of and ability to treat cancer and other diseases.

Therapeutic Targeting of MERTK and BCL-2 in T-Cell and Early T-Precursor Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) accounts for 15% of childhood ALL. The early T-precursor (ETP-ALL) subset is characterized by an immature T-cell phenotype, chemoresistance, and high rates of induction failure. MERTK receptor tyrosine kinase is ectopically expressed in half of T-ALLs, particularly those with an immature T-cell phenotype, suggesting a role in ETP-ALL. The anti-apoptotic protein B-cell lymphoma-2 (BCL-2) is essential for ETP-ALL cell survival. Here, we show that MERTK and BCL-2 mRNA and protein are preferentially expressed in ETP-ALL patient samples. The dual MERTK/FLT3 inhibitor MRX-2843 decreased MERTK activation and downstream signaling, inhibited cell expansion, and induced cell death in ETP-ALL cell lines. Further, 54% (21/39) of primary T-ALL patient samples were sensitive to MERTK inhibition. Treatment with MRX-2843 significantly reduced leukemia burden and prolonged survival in cell-line-derived T-ALL and ETP-ALL xenograft models. In a patient-derived ETP-ALL xenograft model, treatment with MRX-2843 markedly reduced peripheral blood leukemia and spleen weight compared to vehicle-treated mice and prolonged survival. MRX-2843 also synergized with venetoclax to provide enhanced anti-leukemia activity in ETP-ALL cell cultures, with a dose ratio of 1:20 MRX-2843:venetoclax providing optimal synergy. These data demonstrate the therapeutic potential of MRX-2843 in patients with T-ALL and provide rationale for clinical development. MRX-2843 monotherapy is currently being tested in patients with relapsed leukemia (NCT04872478). Further, our data indicate that combined MERTK and BCL-2 inhibition may be particularly effective for treatment of ETP-ALL.

B-cell acute lymphoblastic leukemia promotes an immune suppressive microenvironment that can be overcome by IL-12.

Immunotherapies have revolutionized the treatment of B-cell acute lymphoblastic leukemia (B-ALL), but the duration of responses is still sub-optimal. We sought to identify mechanisms of immune suppression in B-ALL and strategies to overcome them. Plasma collected from children with B-ALL with measurable residual disease after induction chemotherapy showed differential cytokine expression, particularly IL-7, while single-cell RNA-sequencing revealed the expression of genes associated with immune exhaustion in immune cell subsets. We also found that the supernatant of leukemia cells suppressed T-cell function ex vivo. Modeling B-ALL in mice, we observed an altered tumor immune microenvironment, including compromised activation of T-cells and dendritic cells (DC). However, recombinant IL-12 (rIL-12) treatment of mice with B-ALL restored the levels of several pro-inflammatory cytokines and chemokines in the bone marrow and increased the number of splenic and bone marrow resident T-cells and DCs. RNA-sequencing of T-cells isolated from vehicle and rIL-12 treated mice with B-ALL revealed that the leukemia-induced increase in genes associated with exhaustion, including Lag3, Tigit, and Il10, was abrogated with rIL-12 treatment. In addition, the cytolytic capacity of T-cells co-cultured with B-ALL cells was enhanced when IL-12 and blinatumomab treatments were combined. Overall, these results demonstrate that the leukemia immune suppressive microenvironment can be restored with rIL-12 treatment which has direct therapeutic implications.

Engineered Cytokines for Cancer and Autoimmune Disease Immunotherapy.

Cytokine signaling is critical to a range of biological processes including cell development, tissue repair, aging, and immunity. In addition to acting as key signal mediators of the immune system, cytokines can also serve as potent immunotherapies with more than 20 recombinant products currently Food and Drug Administration (FDA)-approved to treat conditions including hepatitis, multiple sclerosis, arthritis, and various cancers. Yet despite their biological importance and clinical utility, cytokine immunotherapies suffer from intrinsic challenges that limit their therapeutic potential including poor circulation, systemic toxicity, and low tissue- or cell-specificity. In the past decade in particular, methods have been devised to engineer cytokines in order to overcome such challenges and here, the myriad strategies are reviewed that may be employed in order to improve the therapeutic potential of cytokine and chemokine immunotherapies with applications in cancer and autoimmune disease therapy, as well as tissue engineering and regenerative medicine. For clarity, these strategies are collected and presented as they vary across size scales, ranging from single amino acid substitutions, to larger protein-polymer conjugates, nano/micrometer-scale particles, and macroscale implants. Together, this work aims to provide readers with a timely view of the field of cytokine engineering with an emphasis on early-stage therapeutic approaches.

Rational design of multistage drug delivery vehicles for pulmonary RNA interference therapy.

Small interfering RNA (siRNA) therapy has significant potential for the treatment of myriad diseases, including cancer. While intravenous routes of delivery have been found to be effective for efficient targeting to the liver, achieving high accumulations selectively in other organs, including lung tissues, can be a challenge. We demonstrate the rational design and engineering of a layer-by-layer (LbL) nanoparticle-containing aerosol that is able to achieve efficient, multistage delivery of siRNA in vitro. For the purpose, LbL nanoparticles were, for the first time, encapsulated in composite porous micro scale particles using a supercritical CO2-assisted spray drying (SASD) apparatus using chitosan as an excipient. Such particles exhibited aerodynamic properties highly favorable for pulmonary administration, and effective silencing of mutant KRAS in lung cancer cells derived from tumors of a non-small cell lung cancer (NSCLC) autochthonous model. Furthermore, efficient alveolar accumulation following inhalation in healthy mice was also observed, corroborating in vitro aerodynamic results, and opening new perspectives for further studies of effective lung therapies These results show that multistage aerosols assembled by supercritical CO2-assisted spray drying can enable efficient RNA interference therapy of pulmonary diseases including lung cancer.

Enhancing chemotherapy response through augmented synthetic lethality by co-targeting nucleotide excision repair and cell-cycle checkpoints.

In response to DNA damage, a synthetic lethal relationship exists between the cell cycle checkpoint kinase MK2 and the tumor suppressor p53. Here, we describe the concept of augmented synthetic lethality (ASL): depletion of a third gene product enhances a pre-existing synthetic lethal combination. We show that loss of the DNA repair protein XPA markedly augments the synthetic lethality between MK2 and p53, enhancing anti-tumor responses alone and in combination with cisplatin chemotherapy. Delivery of siRNA-peptide nanoplexes co-targeting MK2 and XPA to pre-existing p53-deficient tumors in a highly aggressive, immunocompetent mouse model of lung adenocarcinoma improves long-term survival and cisplatin response beyond those of the synthetic lethal p53 mutant/MK2 combination alone. These findings establish a mechanism for co-targeting DNA damage-induced cell cycle checkpoints in combination with repair of cisplatin-DNA lesions in vivo using RNAi nanocarriers, and motivate further exploration of ASL as a generalized strategy to improve cancer treatment.

Rapid Assembly and Screening of Multivalent Immune Cell-Redirecting Therapies for Leukemia.

Therapies that bind with immune cells and redirect their cytotoxic activity toward diseased cells represent a promising and versatile approach to immunotherapy with applications in cancer, lupus, and other diseases; traditional methods for discovering these therapies, however, are often time-intensive and lack the throughput of related target-based discovery approaches. Inspired by the observation that the cytokine, IL-12, can enhance antileukemic activity of the clinically approved T cell redirecting therapy, blinatumomab, here we describe the structure and assembly of a chimeric immune cell-redirecting agent which redirects the lytic activity of primary human T cells toward leukemic B cells and simultaneously cotargets the delivery of T cell-stimulating IL-12. We further describe a novel method for the parallel assembly of compositionally diverse libraries of these bispecific T cell engaging cytokines (BiTEokines) and their high-throughput phenotypic screening, requiring just days for hit identification and the analysis of composition-function relationships. Using this approach, we identified CD19 × CD3 × IL12 compounds that exhibit ex vivo lytic activity comparable to current FDA-approved therapies for leukemia and correlated drug treatment with specific cell-cell contact, cytokine delivery, and leukemia cell lysis. Given the modular nature of these multivalent compounds and their rapid assembly/screening, we anticipate facile extension of this therapeutic approach to a wide range of immune cells, diseased cells, and soluble protein combinations in the future.

Optical Control of Cytokine Signaling via Bioinspired, Polymer-Induced Latency.

Cytokine signaling is challenging to study and therapeutically exploit as the effects of these proteins are often pleiotropic. A subset of cytokines can, however, achieve signal specificity via association with latency-inducing proteins, which cage the cytokine until disrupted by discreet biological stimuli. Inspired by this precision, here, we describe a strategy for synthetic induction of cytokine latency via modification with photolabile polymers that mimic latency while attached then restore protein activity in response to light, thus controlling the magnitude, duration, and location of cytokine signals. We characterize the high dynamic range of cytokine activity modulation and find that polymer-induced latency, alone, can prolong in vivo circulation and bias receptor subunit binding. We further show that protein derepression can be achieved with a near single-cell resolution and demonstrate the feasibility of transcutaneous photoactivation. Future extensions of this approach could enable multicolor, optical reprogramming of cytokine signaling networks and more precise immunotherapies.

Tuning Nanoparticle Interactions with Ovarian Cancer through Layer-by-Layer Modification of Surface Chemistry.

Nanoparticle surface chemistry is a fundamental engineering parameter that governs tumor-targeting activity. Electrostatic assembly generates controlled polyelectrolyte complexes through the process of adsorption and charge overcompensation utilizing synthetic polyions and natural biomacromolecules; it can yield films with distinctive hydration, charge, and presentation of functional groups. Here, we used electrostatic layer-by-layer (LbL) assembly to screen 10 different surface chemistries for their ability to preferentially target human ovarian cancer in vitro. Our screen identified that poly-l-aspartate, poly-l-glutamate, and hyaluronate-coated LbL nanoparticles have striking specificity for ovarian cancer, while sulfated poly(ß-cyclodextrin) nanoparticles target noncancerous stromal cells. We validated top candidates for tumor-homing ability with a murine model of metastatic disease and with patient-derived ovarian cancer spheroids. Nanoparticle surface chemistry also influenced subcellular trafficking, indicating strategies to target the cell membrane, caveolae, and perinuclear vesicles. Our results confirm LbL is a powerful tool to systematically engineer nanoparticles and achieve specific targeting.

RNA-Peptide nanoplexes drug DNA damage pathways in high-grade serous ovarian tumors.

DNA damaging chemotherapy is a cornerstone of current front-line treatments for advanced ovarian cancer (OC). Despite the fact that a majority of these patients initially respond to therapy, most will relapse with chemo-resistant disease; therefore, adjuvant treatments that synergize with DNA-damaging chemotherapy could improve treatment outcomes and survival in patients with this deadly disease. Here, we report the development of a nanoscale peptide-nucleic acid complex that facilitates tumor-specific RNA interference therapy to chemosensitize advanced ovarian tumors to frontline platinum/taxane therapy. We found that the nanoplex-mediated silencing of the protein kinase, MK2, profoundly sensitized mouse models of high-grade serous OC to cytotoxic chemotherapy by blocking p38/MK2-dependent cell cycle checkpoint maintenance. Combined RNAi therapy improved overall survival by 37% compared with platinum/taxane chemotherapy alone and decreased metastatic spread to the lungs without observable toxic side effects. These findings suggest (a) that peptide nanoplexes can serve as safe and effective delivery vectors for siRNA and (b) that combined inhibition of MK2 could improve treatment outcomes in patients currently receiving frontline chemotherapy for advanced OC.

Cellular uptake of nanoparticles: journey inside the cell.

Nanoscale materials are increasingly found in consumer goods, electronics, and pharmaceuticals. While these particles interact with the body in myriad ways, their beneficial and/or deleterious effects ultimately arise from interactions at the cellular and subcellular level. Nanoparticles (NPs) can modulate cell fate, induce or prevent mutations, initiate cell-cell communication, and modulate cell structure in a manner dictated largely by phenomena at the nano-bio interface. Recent advances in chemical synthesis have yielded new nanoscale materials with precisely defined biochemical features, and emerging analytical techniques have shed light on nuanced and context-dependent nano-bio interactions within cells. In this review, we provide an objective and comprehensive account of our current understanding of the cellular uptake of NPs and the underlying parameters controlling the nano-cellular interactions, along with the available analytical techniques to follow and track these processes.

Highly scalable, closed-loop synthesis of drug-loaded, layer-by-layer nanoparticles.

Layer-by-layer (LbL) self-assembly is a versatile technique from which multicomponent and stimuli-responsive nanoscale drug carriers can be constructed. Despite the benefits of LbL assembly, the conventional synthetic approach for fabricating LbL nanoparticles requires numerous purification steps that limit scale, yield, efficiency, and potential for clinical translation. In this report, we describe a generalizable method for increasing throughput with LbL assembly by using highly scalable, closed-loop diafiltration to manage intermediate purification steps. This method facilitates highly controlled fabrication of diverse nanoscale LbL formulations smaller than 150 nm composed from solid-polymer, mesoporous silica, and liposomal vesicles. The technique allows for the deposition of a broad range of polyelectrolytes that included native polysaccharides, linear polypeptides, and synthetic polymers. We also explore the cytotoxicity, shelf life and long-term storage of LbL nanoparticles produced using this approach. We find that LbL coated systems can be reliably and rapidly produced: specifically, LbL-modified liposomes could be lyophilized, stored at room temperature, and reconstituted without compromising drug encapsulation or particle stability, thereby facilitating large scale applications. Overall, this report describes an accessible approach that significantly improves the throughput of nanoscale LbL drug-carriers that show low toxicity and are amenable to clinically relevant storage conditions.

Designer Dual Therapy Nanolayered Implant Coatings Eradicate Biofilms and Accelerate Bone Tissue Repair.

Infections associated with orthopedic implants cause increased morbidity and significant healthcare cost. A prolonged and expensive two-stage procedure requiring two surgical steps and a 6-8 week period of joint immobilization exists as today's gold standard for the revision arthroplasty of an infected prosthesis. Because infection is much more common in implant replacement surgeries, these issues greatly impact long-term patient care for a continually growing part of the population. Here, we demonstrate that a single-stage revision using prostheses coated with self-assembled, hydrolytically degradable multilayers that sequentially deliver the antibiotic (gentamicin) and the osteoinductive growth factor (BMP-2) in a time-staggered manner enables both eradication of established biofilms and complete and rapid bone tissue repair around the implant in rats with induced osteomyelitis. The nanolayered construct allows precise independent control of release kinetics and loading for each therapeutic agent in an infected implant environment. Antibiotics contained in top layers can be tuned to provide a rapid release at early times sufficient to eliminate infection, followed by sustained release for several weeks, and the underlying BMP-2 component enables a long-term sustained release of BMP-2, which induced more significant and mechanically competent bone formation than a short-term burst release. The successful growth factor-mediated osteointegration of the multilayered implants with the host tissue improved bone-implant interfacial strength 15-fold when compared with the uncoated one. These findings demonstrate the potential of this layered release strategy to introduce a durable next-generation implant solution, ultimately an important step forward to future large animal models toward the clinic.

Engineering nanolayered particles for modular drug delivery.

Layer-by-layer (LbL) based self-assembly of nanoparticles is an emerging and powerful method to develop multifunctional and tissue responsive nanomedicines for a broad range of diseases. This unique assembly technique is able to confer a high degree of modularity, versatility, and compositional heterogeneity to nanoparticles via the sequential deposition of alternately charged polyelectrolytes onto a colloidal template. LbL assembly can provide added functionality by directly incorporating a range of functional materials within the multilayers including nucleic acids, synthetic polymers, polypeptides, polysaccharides, and functional proteins. These materials can be used to generate hierarchically complex, heterogeneous thin films on an extensive range of both traditional and novel nanoscale colloidal templates, providing the opportunity to engineer highly precise systems capable of performing the numerous tasks required for systemic drug delivery. In this review, we will discuss the recent advancements towards the development of LbL nanoparticles for drug delivery and diagnostic applications, with a special emphasis on the incorporation of biostability, active targeting, desirable drug release kinetics, and combination therapies into LbL nanomaterials. In addition to these topics, we will touch upon the next steps for the translation of these systems towards the clinic.