CD22L Conjugation to Insulin Attenuates Insulin-Specific B Cell Activation.

Pancreatic islet-reactive B lymphocytes promote Type 1 diabetes (T1D) by presenting an antigen to islet-destructive T cells. Teplizumab, an anti-CD3 monoclonal, delays T1D onset in patients at risk, but additional therapies are needed to prevent the disease entirely. Therefore, bifunctional molecules were designed to selectively inhibit T1D-promoting anti-insulin B cells by conjugating a ligand for the B cell inhibitory receptor CD22 (i.e., CD22L) to insulin, which permit these molecules to concomitantly bind to anti-insulin B cell receptors (BCRs) and CD22. Two prototypes were synthesized: 2:2 insulin-CD22L conjugate on a 4-arm PEG backbone, and 1:1 insulin-CD22L direct conjugate. Transgenic mice (125TgSD) expressing anti-insulin BCRs provided cells for in vitro testing. Cells were cultured with constructs for 3 days, then assessed by flow cytometry. Duplicate wells with anti-CD40 simulated T cell help. A 2-insulin 4-arm PEG control caused robust proliferation and activation-induced CD86 upregulation. Anti-CD40 further boosted these effects. This may indicate that BCR-cross-linking occurs when antigens are tethered by the PEG backbone as soluble insulin alone has no effect. Addition of CD22L via the 2:2 insulin-CD22L conjugate restored B cell properties to that of controls without an additional beneficial effect. In contrast, the 1:1 insulin-CD22L direct conjugate significantly reduced anti-insulin B cell proliferation in the presence of anti-CD40. CD22L alone had no effect, and the constructs did not affect the WT B cells. Thus, multivalent antigen constructs tend to activate anti-insulin B cells, while monomeric antigen-CD22L conjugates reduce B cell activation in response to simulated T cell help and reduce pathogenic B cell numbers without harming normal cells. Therefore, monomeric antigen-CD22L conjugates warrant futher study and may be promising candidates for preclinical trials to prevent T1D without inducing immunodeficiency.

Multivalent Scaffolds to Promote B cell Tolerance.

Autoimmune diseases are characterized by aberrant immune responses toward self-antigens. Current treatments lack specificity, promoting adverse effects by broadly suppressing the immune system. Therapies that specifically target the immune cells responsible for disease are a compelling strategy to mitigate adverse effects. Multivalent formats that display numerous binding epitopes off a single scaffold may enable selective immunomodulation by eliciting signals through pathways unique to the targeted immune cells. However, the architecture of multivalent immunotherapies can vary widely, and there is limited clinical data with which to evaluate their efficacy. Here, we set forth to review the architectural properties and functional mechanisms afforded by multivalent ligands and evaluate four multivalent scaffolds that address autoimmunity by altering B cell signaling pathways. First, we address both synthetic and natural polymer backbones functionalized with a variety of small molecule, peptide, and protein ligands for probing the effects of valency and costimulation. Then, we review nanoparticles composed entirely from immune signals which have been shown to be efficacious. Lastly, we outline multivalent liposomal nanoparticles capable of displaying high numbers of protein antigens. Taken together, these examples highlight the versatility and desirability of multivalent ligands for immunomodulation and illuminate strengths and weaknesses of multivalent scaffolds for treating autoimmunity.

Novel 3D Force Sensors for a Cost-Effective 3D Force Plate for Biomechanical Analysis.

Three-dimensional force plates are important tools for biomechanics discovery and sports performance practice. However, currently, available 3D force plates lack portability and are often cost-prohibitive. To address this, a recently discovered 3D force sensor technology was used in the fabrication of a prototype force plate. Thirteen participants performed bodyweight and weighted lunges and squats on the prototype force plate and a standard 3D force plate positioned in series to compare forces measured by both force plates and validate the technology. For the lunges, there was excellent agreement between the experimental force plate and the standard force plate in the X-, Y-, and Z-axes (r = 0.950-0.999, p < 0.001). For the squats, there was excellent agreement between the force plates in the Z-axis (r = 0.996, p < 0.001). Across axes and movements, root mean square error (RMSE) ranged from 1.17% to 5.36% between force plates. Although the current prototype force plate is limited in sampling rate, the low RMSEs and extremely high agreement in peak forces provide confidence the novel force sensors have utility in constructing cost-effective and versatile use-case 3D force plates.

Design of a Tumor Binding GMCSF as Intratumoral Immunotherapy of Solid Tumors.

Next-generation cancer immunotherapies may utilize immunostimulants to selectively activate the host immune system against tumor cells. Checkpoint inhibitors (CPIs) like anti-PD1/PDL-1 that inhibit immunosuppression have shown unprecedented success but are only effective in the 20-30% of patients that possess an already "hot" (immunogenic) tumor. In this regard, intratumoral (IT) injection of immunostimulants is a promising approach since they can work synergistically with CPIs to overcome the resistance to immunotherapies by inducing immune stimulation in the tumor. One such immunostimulant is granulocyte macrophage-colony-stimulating factor (GMCSF) that functions by recruiting and activating antigen-presenting cells (dendritic cells) in the tumor, thereby initiating anti-tumor immune responses. However, key problems with GMCSF are lack of efficacy and the risk of systemic toxicity caused by the leakage of GMCSF from the tumor tissue. We have designed tumor-retentive versions of GMCSF that are safe yet potent immunostimulants for the local treatment of solid tumors. The engineered GMCSFs (eGMCSF) were synthesized by recombinantly fusing tumor-ECM (extracellular matrix) binding peptides to GMCSF. The eGMCSFs exhibited enhanced tumor binding and potent immunological activity in vitro and in vivo. Upon IT administration, the tumor-retentive eGMCSFs persisted in the tumor, thereby alleviating systemic toxicity, and elicited localized immune activation to effectively turn an unresponsive immunologically "cold" tumor "hot".

Disrupting N-Glycosylation Using Type I Mannosidase Inhibitors Alters B-Cell Receptor Signaling.

Kifunensine is a known inhibitor of type I α-mannosidase enzymes and has been shown to have therapeutic potential for a variety of diseases and application in the expression of high-mannose N-glycan bearing glycoproteins; however, the compound's hydrophilic nature limits its efficacy. We previously synthesized two hydrophobic acylated derivatives of kifunensine, namely, JDW-II-004 and JDW-II-010, and found that these compounds were over 75-fold more potent than kifunensine. Here we explored the effects of these compounds on different mice and human B cells, and we demonstrate that they affected the cells in a similar fashion to kifunensine, further demonstrating their functional equivalence to kifunensine in assays utilizing primary cells. Specifically, a dose-dependent increase in the formation of high-mannose N-glycans decorated glycoproteins were observed upon treatment with kifunensine, JDW-II-004, and JDW-II-010, but greater potency was observed with the acylated derivatives. Treatment with kifunensine or the acylated derivatives also resulted in impaired B-cell receptor (BCR) signaling of the primary mouse B cells; however, primary human B cells treated with kifunensine or JDW-II-004 did not affect BCR signaling, while a modest increase in BCR signaling was observed upon treatment with JDW-010. Nevertheless, these findings demonstrate that the hydrophobic acylated derivatives of kifunensine can help overcome the mass-transfer limitations of the parent compound, and they may have applications for the treatment of ERAD-related diseases or prove to be more cost-effective alternatives for the generation and production of high-mannose N-glycan bearing glycoproteins.

Glatiramer Acetate Complexed with CpG as Intratumoral Immunotherapy in Combination with Anti-PD-1.

CpG oligodeoxynucleotides are toll-like receptor 9 agonists capable of inducing potent pro-inflammatory immune responses. Although CpG oligodeoxynucleotides have shown promising antitumor effects, their systemic activity can trigger immune-related toxicity, limiting therapeutic application. We previously identified glatiramer acetate (GA), a cationic polypeptide approved for the treatment of relapsing-remitting multiple sclerosis, as an intratumoral delivery agent capable of complexing with CpG, thereby pinning it to the injection site and limiting systemic exposure. Here, we investigated whether the combination of CpG or GA-CpG polyplexes and intraperitoneal anti-PD-1 therapy would result in synergistic efficacy in AT84 and CT26 murine syngeneic models of head and neck and colon cancers, respectively. In both AT84 and CT26 tumor models, intratumoral CpG or GA-CpG treatment similarly suppressed tumor growth, but the efficacy was not amplified with anti-PD-1. Nevertheless, combination treatment increased cytotoxic T cell, helper T cell, and natural killer cell infiltration into AT84 tumors. Surprisingly, the combination of intratumoral GA and intraperitoneal anti-PD-1 treatment resulted in elevated systemic GM-CSF and IL-2 cytokine levels and demonstrated synergistic antitumor effects in the CT26 mouse tumor model. Moreover, tumors that responded most significantly to anti-PD-1 plus GA treatment showed increased markers of infiltration of CD4+ T cells and natural killer cells. Combinations of intratumoral GA or GA-CpG polyplexes with anti-PD-1 treatment warrant further investigation as combination cancer immunotherapy strategies.

Strategies to Screen Anti-AQP4 Antibodies from Yeast Surface Display Libraries.

A rapid and effective method to identify disease-specific antibodies from clinical patients is important for understanding autoimmune diseases and for the development of effective disease therapies. In neuromyelitis optica (NMO), the identification of antibodies targeting the aquaporin-4 (AQP4) membrane protein traditionally involves the labor-intensive and time-consuming process of single B-cell sorting, followed by antibody cloning, expression, purification, and analysis for anti-AQP4 activity. To accelerate patient-specific antibody discovery, we compared two unique approaches for screening anti-AQP4 antibodies from yeast antibody surface display libraries. Our first approach, cell-based biopanning, has strong advantages for its cell-based display of native membrane-bound AQP4 antigens and is inexpensive and simple to perform. Our second approach, FACS screening using solubilized AQP4 antigens, permits real-time population analysis and precision sorting for specific antibody binding parameters. We found that both cell-based biopanning and FACS screening were effective for the enrichment of AQP4-binding clones. These screening techniques will enable library-scale functional interrogation of large natively paired antibody libraries for comprehensive analysis of anti-AQP4 antibodies in clinical samples and for robust therapeutic discovery campaigns.

Remote-Controlled 3D Porous Magnetic Interface toward High-Throughput Dynamic 3D Cell Culture.

Mechanical stimuli have been shown to play a large role in cellular behavior, including cellular growth, differentiation, morphology, homeostasis, and disease. Therefore, developing bioreactor systems that can create complex mechanical environments for both tissue engineering and disease modeling drug screening is appealing. However, many of existing systems are restricted because of their bulky size with external force generators, destructive microenvironment control, and low throughput. These shortcomings have preceded to the utilization of magnetic stimuli responsive materials, given their untethered, fast, and tunable actuation potential at both the microscale and macroscale level, for seamless integration into cell culture wells and microfluidic systems. Nevertheless, magnetic soft materials for cell culture have been limited due to the inability to develop well-defined 3D structures for more complex and physiological relevant mechanical actuation. Herein, we introduce a facile fabrication process to develop magnetic-PDMS (polydimethylsiloxane) porous composite designs with both well-defined and controllable microlevel and macrolevel features to dynamically manipulate 3D cell-laden gel at the scale. The intrinsic stiffness of the magnetic-PDMS porous composites is also modulated to control the deformation potential to mimic physiological relevant strain levels, with 2.89-11% observed in magnetic actuation studies. High cell viability was achieved with the culturing of both human adipose stem cells (hADMSCs) and human umbilical cord mesenchymal stem cells (hUCMSCs) in 3D cell-laden gel interfaced with the magnetic-PDMS porous composite. Also, the highly interconnected porous network of the magnetic-PDMS composites facilitated free diffusion throughout the porous structure showcasing the potential of a multisurface contact 3D porous magnetic structure in both reservoir and 96-well plate insert designs for more complex dynamic mechanical actuation. In conclusion, these studies provide a means for establishing a biocompatible, tunable magnetic-PDMS porous composite with fast and programmable dynamic strain potential making it a suitable platform for high-throughput, dynamic 3D cell culture.

Glatiramer acetate enhances tumor retention and innate activation of immunostimulants.

Cancer immunotherapy aims to stimulate immune cells to recognize and attack tumor tissue. The immunostimulatory polyanions polyI:C and CpG induce potent pro-inflammatory immune responses as TLR3 and TLR9 agonists, respectively. Clinical trials of TLR agonists, however, have been fraught with immune-related adverse events, even when injecting intratumorally in an effort to minimize systemic exposure. We identified Glatiramer Acetate (GA), a positively-charged polypeptide approved for multiple sclerosis, as a delivery agent capable of complexing with polyI:C or CpG and reducing the mobility of these actives. Small nanoparticles termed polyplexes form when mixing positively-charged GA and negatively-charged immunostimulant (polyI:C or CpG). The ratio of GA to immunostimulant directly affected the potency of TLR activation and the mobility of these actives in simulated tumor tissue. Polyplexes of GA and CpG were injected intratumorally in a tumor model of head and neck cancer (HNC) and significantly mitigated tumor growth as compared to the vehicle controls. Intratumoral injections of CpG showed the slowest tumor growth but exhibited dramatically higher systemic proinflammatory cytokine levels compared to polyplexes of GA with CpG. Sequencing of RNA from resected tumors revealed a similar pattern of upregulated proinflammatory cytokines for CpG and polyplexes, a finding supported by histological tumor staining showing similar infiltration of immune cells induced by these treatments. Intratumoral administration of polyplexes of GA with immunostimulant represents a translational approach to enhance local immune responses while mitigating systemic immune-related adverse events.

The era of gene therapy: From preclinical development to clinical application.

Three decades of promise have culminated in the development of gene therapies that can be applied to a broad range of human diseases. After a brief history, we provide an overview of gene therapy types and delivery methods, gene editing technologies, regulatory affairs, clinical trials, approved products, ongoing challenges, and future goals. Information on clinical trials of candidates and on approved products for gene therapy developed between 1988 and 2020 is systematically collated. To obtain this global information, we scanned and reviewed more than 46,000 records of clinical trials from 17 clinical trial database providers. The medical benefits of transformative gene therapies are gradually being accepted by payors, and a significant increase in the number of gene therapy clinical trials and approved gene therapy products has resulted.

Brain Homogenate Decoys for Antigen-Specific Cell Amplification.

Multiple sclerosis is complex and heterogeneous. Better tools are needed to be able to monitor this disease among individuals, but blood-based biomarkers are often too rare to profile. In this work, we developed antigen-specific biomaterials to replicate the central nervous system niche where multiple sclerosis biomarkers are amplified. We incorporated mouse brain homogenate into a microporous gelatin methacrylate network. Homogenate-containing biomaterials differentially stimulated cells and led to the marked amplification of disease-relevant, antigen-specific B cells. These results demonstrate that biomaterials containing primary tissue homogenate retain antigen specificity and may be a useful tool for decoding human autoimmunity.

Remote Sensing and Remote Actuation via Silicone-Magnetic Nanorod Composites.

The capacity for a soft material to combine remote sensing and remote actuation is highly desirable for many applications in soft robotics and wearable technologies. This work presents a silicone elastomer with a suspension of a small weight fraction of ferromagnetic nickel nanorods, which is capable of both sensing deformation and altering stiffness in the presence of an external magnetic field. Cylinders composed of silicone elastomer and 1% by weight nickel nanorods experience large increases in compressive modulus when exposed to an external magnetic field. Incremental compressions totaling 600 g of force applied to the same silicone-nanorod composites increase the magnetic field strength measured by a Hall effect sensor enabling the material to be used as a soft load cell capable of detecting the rate, duration, and magnitude of force applied. In addition, lattice structures are 3D printed using an ink composed of silicone elastomer and 1% by weight nickel nanorods, which possess the same sensing capacity.

Optimized Production of Fc Fusion Proteins by Sortase Enzymatic Ligation.

Fc fusions are a growing class of drugs comprising an antibody Fc domain covalently linked to a protein or peptide and can pose manufacturing challenges. In this study we evaluated three synthetic approaches to generate Fc fusions, using Fc-insulin as a model drug candidate. Engineered human IgG1 was digested with HRV3C to produce an Fc fragment with a C-terminal sortase tag (Fc-LPETGGH6). The synthesis of Fc-insulin2 from Fc-LPETGGH6 was evaluated with direct sortase-mediated ligation (SML) and two chemoenzymatic strategies. Direct SML was performed with triglycine-insulin, and chemoenzymatic strategies used to SML fuse either triglycine-azide or triglycine-DBCO prior to linking insulin with copper-catalyzed or strain-promoted azidealkyne cycloaddition. Reaction conditions were optimized by evaluating reagent concentrations, relative equivalents, temperature, and time. Direct SML provided the most effective reaction yields, converting 60-70% of Fc-LPETGGH6 to Fc-insulin2, whereas our optimized chemoenzymatic synthesis converted 30-40% of Fc-LPETGGH6 to Fc-insulin2. Here we show that SML is a practical and efficient method to synthesize Fc fusions and provide an optimized pathway for fusion drug synthesis.

Tetrameric Fluorescent Antigen Arrays for Single-Step Identification of Antigen-Specific B Cells.

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and the use of four-arm, fluorescent PEG-antigen conjugates to selectively identify antigen-specific B cells through avid engagement with cognate B cell receptors. Using modular click chemistry and commercially available fluorophore kit chemistries, we demonstrated the versatility of preparing customized fluorescent PEG-conjugates by creating distinct arrays for proteolipid protein (PLP139-151) and insulin, which are important autoantigens in murine models of multiple sclerosis and type 1 diabetes, respectively. Assays were developed for each fluorescent conjugate in its respective disease model using flow cytometry. Antigen arrays were compared to monovalent autoantigen to quantify the benefit of multimerization onto PEG backbones. Finally, we illustrated the utility of this platform by isolating and assessing anti-insulin B cell responses after antigen stimulation ex vivo. Labeling insulin-specific B cells enabled the amplified detection of changes to co-stimulation (CD86) that were otherwise dampened in aggregate B cell analysis. Together, this report enables the production and use of fluorescent antigen arrays as a robust tool for probing B cell populations.

Autoantigen Tetramer Silences Autoreactive B Cell Populations.

Many autoimmune therapies focus on immune suppression to reduce symptom severity and halt disease progression; however, currently approved treatments lack specificity for the autoantigen and rely on more global immune suppression. Multivalent antigen arrays can disarm pathogenic autoimmune B cell populations that specifically recognize the antigen of interest via their B cell receptor (BCR). Disarmament may be achieved by BCR engagement, cross-linking, and sustained receptor occupancy as a result of multivalent, high avidity BCR binding. To engage and explore this mechanism, a tetramer display of the encephalogenic proteolipid peptide (PLP139-151), referred to as 4-arm PLP139-151, was synthesized by copper-catalyzed azide-alkyne cycloaddition chemistry. Subcutaneous administration of 4-arm PLP139-151 completely ameliorated symptoms of paralysis in a mouse model of multiple sclerosis known as experimental autoimmune encephalomyelitis. Competitive binding of 4-arm PLP139-151 to PLP139-151-specific IgG in the mouse serum demonstrated the enhanced avidity associated with the multivalent array compared to the free peptide. Furthermore, key PLP139-151-reactive B cells were depleted following 4-arm PLP139-151 treatment, resulting in significant reduction of proinflammatory cytokines. Together, these data demonstrate the potential of 4-arm PLP139-151 to silence autoreactive B cell populations and limit the downstream activation of effector cells.

Constructing a Biomaterial to Simulate Extracellular Drug Transport in Solid Tumors.

Designing an in vitro model of the tumor extracellular microenvironment to screen intratumoral drugs is an active challenge. As recent clinical successes of human intratumoral therapies stimulate research on intratumoral delivery, a need for a 3D tumor model to screen intratumoral therapies arises. When injecting the drug formulation directly into the tumor, the biophysics affecting intratumoral retention must be considered; especially for biologic therapies, which may be dominated by extracellular transport mechanisms. Fibrotic regions in solid tumors are typically rich in collagen I fibers. Using shear rheology, head and neck tumors with higher collagen density show a higher stiffness. Similarly, the stiffness of the hyaluronic acid (HA) hydrogel models is increased by adding collagen fibers to model the bulk biomechanical properties of solid tumors. HA hydrogels are then used as intratumoral injection site simulators to model in vitro the retention of glatiramer acetate (GA) and polyethylene glycol (PEG) administered intratumorally. Both compounds are also injected in murine tumors and retention is studied ex vivo for comparison. Retention of GA in the hydrogels is significantly longer than PEG, analogous to the solid tumors, suggesting the utility of HA hydrogels with collagen I fibers for screening extracellular drug transport after intratumoral administration.

Human intratumoral therapy: Linking drug properties and tumor transport of drugs in clinical trials.

Cancer therapies aim to kill tumor cells directly or engage the immune system to fight malignancy. Checkpoint inhibitors, oncolytic viruses, cell-based immunotherapies, cytokines, and adjuvants have been applied to prompt the immune system to recognize and attack cancer cells. However, systemic exposure of cancer therapies can induce unwanted adverse events. Intratumoral administration of potent therapies utilizes small amounts of drugs, in an effort to minimize systemic exposure and off-target toxicities. Here, we discuss the properties of the tumor microenvironment and transport considerations for intratumoral drug delivery. Specifically, we consider various tumor tissue factors and physicochemical factors that can affect tumor retention after intratumoral injection. We also review approved and clinical-stage intratumoral therapies and consider how the molecular and biophysical properties (e.g. size and charge) of these therapies influences intratumoral transport (e.g. tumor retention and cellular uptake). Finally, we offer a critical review and highlight several emerging approaches to promote tumor retention and limit systemic exposure of potent intratumoral therapies.

Immunostimulant Complexed With Polylysine Limits Transport and Maintains Immune Cell Activation.

Activation of the immune system to treat cancer has emerged as a powerful therapy tool, however, treatments must overcome the immunosuppressive microenvironment established by tumors. Toll-like receptor (TLR) agonists like CpG and polyI:C are potent stimulators of non-specific, pro-inflammatory immune responses, targeting TLR9 and TLR3, respectively. While these immunostimulants seem promising, systemic exposure can eventually induce severe side effects. Adverse inflammatory reactions in healthy tissues may be avoided by delivering and retaining immunostimulants in proximity to tumors or to primary sites of tumor metastases. Immunostimulants such as CpG and polyI:C cannot be completely immobilized, however, since the target TLR9 and TLR3 are located intracellularly. Previously, polycations like poly-l-lysine (PLL) have been complexed to the anionic CpG or polyI:C with the purpose of improving intracellular delivery and potency. Here, the relationship between PLL molecular weight and immunostimulant complexation, TLR activation, and transport in a simple, model tumor microenvironment was investigated. The polyplexes could be formulated to dramatically limit immunostimulant transport suggesting the potential for injection site retention and minimized systemic exposure of immunostimulants. The molecular weight of PLL and ratio of PLL to immunostimulant affected the accessibility of the immunostimulant within the polyplex and polyplex interaction strength.

Linking autoantigen properties to mechanisms of immunity.

Antigen-specific immunotherapies (ASIT) present compelling potential for introducing precision to the treatment of autoimmune diseases where nonspecific, global immunosuppression is currently the only treatment option. Central to ASIT design is the delivery of autoantigen, which parallels allergy desensitization approaches. Clinical success in tolerizing allergen-specific responses spans longer than a century, but autoimmune ASITs have yet to see an FDA-approved breakthrough. Allergens and autoantigens differ substantially in physicochemical properties, and these discrepancies influence the nature of their interactions with the immune system. Approved allergen-specific immunotherapies are typically administered as water soluble, neutrally charged protein fractions from 10 to 70 kDa. Conversely, autoantigens are native proteins that exhibit wide-ranging sizes, solubilities, and charges that render them susceptible to immunogenicity. To translate the success of allergen hyposensitization to ASIT, delivery strategies may be necessary to effectively format autoantigens, guide biodistribution, and engage appropriate immune mechanisms.

Development of lipid membrane based assays to accurately predict the transfection efficiency of cell-penetrating peptide-based gene nanoparticles.

Successful gene therapy requires the development of vectors that enable efficient delivery of genetic materials (e.g., pDNA or siRNA) to targeted cells, without degradation of the genetic materials. We have shown that nanoparticles formed by combining cell-penetrating peptide and pDNA (CPP-pDNA) into complexes and condensing them with calcium chloride can provide gene nanoparticles with high transfection efficiency and low cytotoxicity. In this work, we compare in situ measurements of the membrane insertion potential of three arginine-based gene nanoparticles (RW9-NPs, R9-NPs, and RH9-NPs) using four lipid compositions and two types of model membrane (Langmuir monolayers vs. supported bilayers) with their transfection efficiency in two human cancer cell lines. Using a Langmuir trough, we measured the membrane insertion potential of our gene nanoparticles to model membrane monolayers. A Quartz Crystal Microbalance with Dissipation (QCM-D) technique was used to monitor the adsorption of these nanoparticles to lipid bilayers of various compositions. Finally, gene expression using these nanoparticles was measured in breast cancer and cervical cancer cell lines. Our cell culture studies indicate that although R9-NPs and RW9-NPs show a significant increase in transfection efficiency compared to free pDNA, RH9-NPs do not show any significant difference. Both the Langmuir monolayer and QCM-D bilayer studies show that these results are best reflected in the in situ measurement assays when lipid systems containing a mixture of phospholipids, cholesterol, and sphingolipids are used. It is important to note that the mechanism of penetration is expected to differ for RW9 vs. R9; however, gene nanoparticles containing either of these CPPs show similar transfection efficiency. Our results therefore demonstrate that the design of predictive assays for gene therapy using CPPs must involve carefully chosen model lipid membrane systems that accurately represent the varying compositions of cell membranes.