Stability of ranibizumab during continuous delivery from the Port Delivery Platform.

The Port Delivery System with ranibizumab (PDS) is an innovative intraocular drug delivery system that has the potential to reduce treatment burden in patients with retinovascular diseases. The Port Delivery Platform (PD-P) implant is a permanent, indwelling device that can be refilled in situ through a self-sealing septum and is designed to continuously deliver ranibizumab by passive diffusion through a porous titanium release control element. We present results for the studies carried out to characterize the stability of ranibizumab for use with the PD-P. Simulated administration, in vitro release studies, and modeling studies were performed to evaluate the compatibility of ranibizumab with the PD-P administration components, and degradation and photostability in the implant. Simulated administration studies demonstrated that ranibizumab was highly compatible with the PD-P administration components (initial fill and refill needles) and commercially available administration components (syringe, transfer needle, syringe closure). Subsequent simulated in vitro release studies examining continuous delivery for up to 12 months in phosphate buffered saline, a surrogate for human vitreous, showed that the primary degradation products of ranibizumab were acidic variants. The presence of these variants increased over time and potency remained high. The stability attributes of ranibizumab were consistent across multiple implant refill-exchanges. Despite some degradation within the implant, the absolute mass of variants released daily from the implant was low due to the continuous release mechanism of the implant. Simulated light exposure within the implant resulted in small increases in the relative amount of ranibizumab degradants compared with those seen over 6 months.

Hydroxypropyl methyl cellulose derivatives stabilize fragment antibody against aggregation in spray dried formulations at elevated temperature and resist pH changes.

The ability to deliver stable and active dried protein therapeutics from biopharmaceutical drug delivery systems is critical for solid dosage formulation development. Spray dried formulations with carefully selected excipients provide a unique opportunity in amorphous phase stabilization of the therapeutic proteins. Herein, we discuss the role of hydroxypropyl methylcellulose acetate succinate (HPMCAS) derivatives as polymeric excipients for stabilizing a model fragment antibody (Fab2) during high temperature processing and in possible low pH environments of a drug delivery platform. The effects of high temperature processing and microenvironmental pH sensitivity are of particular interest to us due to their adverse impact on stability of molecules that demonstrate temperature and pH dependent inactivation within drug delivery devices. It appears in solid state at 90 °C and 37 °C and within low pH micro-environment HPMCAS protects protein against aggregation. The high temperature performance of HPMCAS is comparable to that of a disaccharide excipient like trehalose in spray dried protein powder. Simultaneously, inside a poly(lactic-co-glycolic acid) (PLGA) based delivery system HPMCAS provides protection to a pH sensitive protein against acidic degradation products from aqueous hydrolysis of PLGA.

Design of PLGA-Based Drug Delivery Systems Using a Physically-Based Sustained Release Model.

An extensive data set has been developed and used to further the progress of a model-informed design of controlled drug release. An improved drug-release model with mechanistic modeling of hydrolytic polymer degradation is used and validated by comparing model predictions to in vitro experiments. Combining parameter estimates from the literature with model fits to the data set, this study can aid in achieving a priori design of controlled drug release from a model PLGA release system. A systematic series of model release systems were formulated with FITC-labeled dextran, as a surrogate for biopharmaceuticals, in PLGA rods over a broad range of compositions. While general comparisons between the model and experiments were favorable, important discrepancies were identified for several formulations with significant first-phase drug release. Supported by cross-sectional fluorescence microscopy images of the FITC-dextran distribution within the rods, this first-phase release was attributed to a combination of two main factors: (1) percolation of the drug particles and (2) swelling of and pore formation in the rods due to water uptake. These observations indicate the importance of careful selection of the PLGA polymer grade when designing drug release systems but also reflect a need for better understanding of phenomena such as pore formation. Adapting model parameters, without modifying the physical processes included in the model, enabled accurate fitting of the experimental data for all formulations, highlighting the applicability of the model.

Release Mechanisms and Practical Percolation Threshold for Long-acting Biodegradable Implants: An Image to Simulation Study.

The development of long-acting drug formulations requires efficient characterization technique as the designed 6-12 months release duration renders real-time in vitro and in vivo experiments cost and time prohibitive. Using a novel image-based release modeling method, release profiles were predicted from X-Ray Microscopy (XRM) of T0 samples. A validation study with the in vitro release test shows good prediction accuracy of the initial burst release. Through fast T0 image-based release prediction, the impact of formulation and process parameters on burst release rate was investigated. Recognizing the limitations of XRM, correlative imaging with Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) was introduced. A water stress test was designed to directly elucidate the formation of pores through polymer-drug-water interplay. Through an iterative correction method that considers poly(lactic-co-glycolic acid) (PLGA) polymer degradation, good agreement was achieved between release predictions  using FIB-SEM images acquired from T0 samples and in vitro testing data. Furthermore, using image-based release simulations, a practical percolation threshold was identified that has profound influence on the implant performance.  It is proposed as an important critical quality attribute for biodegradable long-acting delivery system, that needs to be investigated and quantified.

Microstructure, Quality, and Release Performance Characterization of Long-Acting Polymer Implant Formulations with X-Ray Microscopy and Quantitative AI Analytics.

Long-acting implants are typically formulated using carrier(s) with specific physical and chemical properties, along with the active pharmaceutical ingredient (API), to achieve the desired daily exposure for the target duration of action. In characterizing such formulations, real-time in-vitro and in-vivo experiments that are typically used to characterize implants are lengthy, costly, and labor intensive as these implants are designed to be long acting. A novel characterization technique, combining high resolution three-dimensional X-Ray microscopy imaging, image-based quantification, and transport simulation, has been employed to provide a mechanistic understanding of formulation and process impact on the microstructures and performance of a polymer-based implant. Artificial intelligence-based image segmentation and image data analytics were used to convert morphological features visualized at high resolution into numerical microstructure models. These digital models were then used to calculate key physical parameters governing drug transport in a polymer matrix, including API uniformity, API domain size, and permeability. This powerful new tool has the potential to advance the mechanistic understanding of the interplay between drug-microstructure and performance and accelerate the therapeutic development long-acting implants.

Accelerated in vitro release testing method for a long-acting peptide-PLGA formulation.

Poly (lactic-co-glycolic acid) (PLGA), a biocompatible and biodegradable polymer, is one of the most commonly used vehicles for controlled-release (CR) implantable dosage forms. Drug molecules formulated in such CR vehicles are released slowly over an extended period of time - often months to years - posing challenges for batch release and quality control testing. Thus, reliable and reproducible accelerated testing methods are required to bridge this gap during early formulation development. This work describes the development of an accelerated in vitro release testing method to predict the real-time in vitro release of a synthetic peptide from a 6-month CR PLGA implant formulation. While accelerated methods have been previously reported for PLGA-based formulations, this work describes a unique case of an aggregation-prone peptide, which required careful attention to the impact of different conditions on both release kinetics and peptide stability. This method describes a suitable combination of release conditions that could help in understanding the release profiles of such peptides prone to aggregation. Parameters including pH, buffer species, temperature, and addition of organic co-solvents and surfactants were evaluated separately and in combination for their ability to achieve complete peptide release within 2 weeks while accurately recapitulating release rate, profile and peptide stability. The accelerated release method that gave the best agreement with real-time release was a mixed media of co-solvent (5% tetrahydrofuran), surfactant (5% TritonX-100) and elevated temperature (50 °C) in a neutral buffer (PBS pH 7.4). This optimized accelerated release method achieved complete release of the peptide load within 14-21 days compared to 3- to 6-months of real-time release and could discriminate critical differences in release behavior between different CR formulations to guide formulation and process development.

Discovery of a dual pathway aggregation mechanism for a therapeutic constrained peptide.

Constrained peptides (CPs) have emerged as attractive candidates for drug discovery and development. To fully unlock the therapeutic potential of CPs, it is crucial to understand their physical stability and minimize the formation of aggregates that could induce immune responses. Although amyloid like aggregates have been researched extensively, few studies have focused on aggregates from other peptide scaffolds (e.g., CPs). In this work, a streamlined approach to effectively profile the nature and formation pathway of CP aggregates was demonstrated. Aggregates of various sizes were detected and shown to be amorphous. Though no major changes were found in peptide structure upon aggregation, these aggregates appeared to have mixed natures, consisting of primarily non-covalent aggregates with a low level of covalent species. This co-existence phenomenon was also supported by two kinetic pathways observed in time- and temperature-dependent aggregation studies. Furthermore, a stability study with 8 additional peptide variants exhibited good correlation between aggregation propensity and peptide hydrophobicity. Therefore, a dual aggregation pathway was proposed, with the non-covalent aggregates driven by hydrophobic interactions, whereas the covalent ones formed through disulfide scrambling. Overall, the workflow presented here provides a powerful strategy for comprehensive characterization of peptide aggregates and understanding their mechanisms of formation.

Long-Term Stability of Anti-Vascular Endothelial Growth Factor (a-VEGF) Biologics Under Physiologically Relevant Conditions and Its Impact on the Development of Long-Acting Delivery Systems.

The port delivery system with ranibizumab (PDS) is an investigational long-acting drug delivery system for the continuous release of ranibizumab, an anti-VEGF biologic, in the vitreous humor. The efficacy of the PDS implant relies on the maintenance of long-term drug stability under physiological conditions. Herein, the long-term stability of three anti-VEGF biologics - ranibizumab, bevacizumab and aflibercept - was investigated in phosphate buffered saline (PBS) at 37 °C for several months. Comparison of stability profiles shows that bevacizumab and aflibercept are increasingly prone to aggregation whereas ranibizumab undergoes minimal aggregation. Ranibizumab also shows the smallest loss in antigen binding capacity after long-term incubation in PBS. Even though the aggregated forms of bevacizumab and aflibercept bind to VEGF, the consequences of aggregation on immunogenicity, implant function and efficacy are unknown. These results highlight the importance of maintaining long-term drug stability under physiologically relevant conditions which is necessary for achieving efficacy with an in vivo continuous drug delivery device such as the PDS implant.

Intravitreal Administration of Acetyl Triethyl Citrate and Benzyl Benzoate Is Retinotoxic in Rabbits but Not in Cynomolgus Monkeys.

Sustained drug delivery formulations are developed to reduce dose frequency while maintaining efficacy of intravitreal (ITV) administered therapeutics. Available safety data for components novel to the eye's posterior segment may be limited, requiring preclinical assessments to identify potential toxicities. We evaluated the in vivo and in vitro safety of two solvents, acetyl triethyl citrate (ATEC) and benzyl benzoate (BB), as novel sustained delivery formulations for ITV administration. In vivo tolerability was assessed following ITV administration of ATEC and BB to rabbits and cynomolgus monkeys. In rabbits, ITV solvent administration resulted in moderate to severe retinal toxicity characterized by focal retinal necrosis and/or degeneration, sometimes accompanied by inflammation, with a clear association between the physical presence of the solvent and areas of retinal damage. In contrast, solvent administration in monkeys appeared well tolerated, producing no histologic abnormalities. Toxicity in primary human retinal pigment epithelial cells, characterized by cellular toxicity and mitochondrial injury, corroborated the retinal toxicity in rabbits. In conclusion, ITV solvent depots of ATEC or BB result in chemical and focal retinal toxicity in rabbits, but not monkeys. Additional investigation is needed to demonstrate a sufficient margin of safety prior to use of ATEC or BB in ITV drug products.

Ectoine and Hydroxyectoine Stabilize Antibodies in Spray-Dried Formulations at Elevated Temperature and during a Freeze/Thaw Process.

Maintenance of protein stability during manufacture, storage, and delivery is necessary for the successful development of a drug product. Herein, the utility of two compatible solutes-ectoine and hydroxyectoine-in stabilizing a model protein labeled Fab2 has been investigated. Specifically, the performance of ectoine and hydroxyectoine in stabilizing Fab2 in a spray-dried formulation at elevated temperature and after multiple freeze/thaw cycles has been compared with the performance of a formulation containing trehalose and a formulation containing no excipient as controls. In the solid state at 90 and 37 °C and in freeze concentrate systems, ectoine and hydroxyectoine suppress protein aggregation. Like trehalose, hydroxyectoine also limits N-terminal pyroglutamate formation in Fab2 in the solid state. The extent of protein stabilization is dependent on the excipient concentration in the formulation, but at a 1:1 excipient to protein mass ratio, hydroxyectoine is better than trehalose in stabilizing Fab2. The results presented here suggest that ectoine and hydroxyectoine are effective excipients for stabilizing therapeutic antibodies.

Optimizing the Formulation and Lyophilization Process for a Fragment Antigen Binding (Fab) Protein Using Solid-State Hydrogen-Deuterium Exchange Mass Spectrometry (ssHDX-MS).

Solid-state hydrogen-deuterium exchange with mass spectrometry (ssHDX-MS) was evaluated as an analytical method to rapidly screen and select an optimal lyophilized fragment antigen binding protein (Fab) formulation and the optimal lyophilization cycle. ssHDX-MS in lyophilized Fab formulations, varying in stabilizer type and stabilizer/protein ratio, was conducted under controlled humidity and temperature. The extent of deuterium incorporation was measured using mass spectrometry and correlated with solid-state stress degradation at 50 °C as measured by size exclusion chromatography (SEC) and ion-exchange chromatography (IEC). ssHDX-MS was also used to evaluate the impact of three different types of lyophilization processing on storage stability: controlled ice nucleation (CN), uncontrolled ice nucleation (UCN), and annealing (AN). The extent of deuterium incorporation for different Fab formulations agreed with the order of solid-state stress degradation, with formulations having lower deuterium incorporation showing lower stress-induced degradation (aggregation and charge modifications). For lyophilization processing, no significant effect of ice nucleation was observed in either solid-state stress degradation or in the extent of deuterium incorporation for high concentration Fab formulations (25 mg/mL). In contrast, for low concentration Fab formulations (2.5 mg/mL), solid-state stability from different lyophilization processes correlated with the extent of deuterium incorporation. The order of solid-state degradation (AN < CN < UCN) was the same as the extent of deuterium incorporation on ssHDX-MS (AN < CN < UCN). The extent of deuterium incorporation on ssHDX-MS correlated well with the solid-state stress degradation for different Fab formulations and lyophilization processing methods. Thus, ssHDX-MS can be used to rapidly screen and optimize the formulation and lyophilization process for a lyophilized Fab, reducing the need for time-consuming stress degradation studies.

Data-Driven Development of Predictive Models for Sustained Drug Release.

Mathematical modeling of drug release can aid in the design and development of sustained delivery systems, but the parameter estimation of such models is challenging owing to the nonlinear mathematical structure and complexity and interdependency of the physical processes considered. Highly parameterized models often lead to overfitting, strong parameter correlations, and as a consequence, inaccurate model predictions for systems not explicitly part of the fitting database. Here, we show that an efficient stochastic optimization algorithm can be used not only to find robust estimates of global minima to such complex problems but also to generate metadata that allow quantitative evaluation of parameter sensitivity and correlation, which can be used for further model refinement and development. A practical methodology is described through the analysis of a predictive drug release model on published experimental data sets. The model is then used to design a zeroth-order release profile in an experimental system consisting of an antibody fragment in a poly(lactic-co-glycolic acid) solvent depot, which is validated experimentally. This approach allows rational decision-making when developing new models, selecting models for a specific application, or designing formulations for experimental trials.

Hydrogels for sustained delivery of biologics to the back of the eye.

Hydrogels are water-laden polymer networks that have been used for myriad biological applications. By controlling the chemistry through which a hydrogel is constructed, a wide range of chemical and physical properties can be accessed, making them an attractive class of biomaterials. In this review, we cover the application of hydrogels for sustained delivery of biologics to the back of the eye. In adapting hydrogels to this purpose, success is dependent on careful consideration of material properties, route of administration, means of injection, and control of drug efflux, all of which are addressed. We also provide a perspective on clinical and chemistry, manufacturing and controls (CMC) considerations that are integral to the development of an ocular hydrogel delivery system.

Trehalose Limits Fragment Antibody Aggregation and Influences Charge Variant Formation in Spray-Dried Formulations at Elevated Temperatures.

The preparation of PLGA rods for sustained release applications via a hot-melt extrusion process employs heat and mechanical shear. Understanding protein stability and degradation mechanisms at high temperature in the solid state is therefore important for the preparation of protein-loaded PLGA rods. The stability of a model protein, labeled Fab2, has been investigated in solid-state formulations containing trehalose at elevated temperatures. Spray-dried formulations containing varying levels of trehalose were exposed to temperatures ranging from 90 to 120 °C. Measurement of aggregation and chemical degradation rates suggests that trehalose limits Fab2 degradation in a concentration-dependent manner, but the effect tends to saturate when the mass ratio of trehalose to protein is around 1 in the solid formulation. The Fab2 secondary structure and spray-dried particle morphology were studied using circular dichroism and scanning electron microscopy techniques, respectively. On the basis of temperature and trehalose-dependent aggregation kinetics as well as changes in spray-dried particle morphology, a mechanism is proposed for the trehalose stabilization of proteins in solid state at elevated temperatures. The results reported here suggest that when fragment antibodies in the solid state are formulated with trehalose as excipient, a high temperature process such as hot-melt extrusion can be successfully accomplished with minimal degradation.

Evaluation of the Toxicity of Intravitreally Injected PLGA Microspheres and Rods in Monkeys and Rabbits: Effects of Depot Size on Inflammatory Response.

Purpose: Poly(lactic-co-glycolic) acid (PLGA) inserts have been successfully developed for the treatment of posterior eye disease as a means of reducing injection frequency of intravitreally administered therapeutics. PLGA microspheres are also of interest for the delivery of intravitreal drugs, since they offer the advantage of being easily injected without surgical procedures or large injectors. Methods: In the current study, the toxicity of PLGA microspheres and rods was investigated in nonhuman primates (NHPs) and rabbits. An in vitro assessment of cytokine responses to PLGA in peripheral blood mononuclear cells (PBMCs) and macrophages was also performed. Results: Intravitreal administration of 3, 10, or 12.5 mg/eye of PLGA microspheres in NHPs resulted in a severe immune response characterized by a foreign body response. Follow-up studies in the rabbit confirmed this finding for PLGA microspheres ranging in size from 20 to 100 µm. In contrast, administration of PLGA rod implants with a similar PLGA mass did not elicit a significant immune response. In vitro assays in PBMCs and macrophages confirmed proinflammatory cytokine release upon treatment with PLGA microspheres but not PLGA rods. Conclusions: These data demonstrate a lack of tolerability of PLGA microspheres upon intravitreal injection, and suggest that the size, shape, and/or surface area of PLGA depots are critical attributes in determining ocular toxicity.

Interfacial properties of POPC/GDO liquid crystalline nanoparticles deposited on anionic and cationic silica surfaces.

Reversed lipid liquid crystalline nanoparticles (LCNPs) of the cubic micellar (I2) phase have high potential in drug delivery applications due to their ability to encapsulate both hydrophobic and hydrophilic drug molecules. Their interactions with various interfaces, and the consequences for the particle structure and integrity, are essential considerations in their effectiveness as drug delivery vehicles. Here, we have studied LCNPs formed of equal fractions of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and glycerol dioleate in the presence of different fractions of the stabilizer Polysorbate 80. We have used a combination of ellipsometry, quartz crystal microbalance with dissipation monitoring and neutron reflectometry to reveal the structure and composition of the adsorbed layer on both anionic silica and cationic (aminopropyltriethoxysilane) silanized surfaces. For both types of surfaces, there is a spread near-surface layer comprising lipid and polymer as well as a sparse coverage of intact particles. The composition of the near-surface layer is very close to that of the particles, in contrast to the lipid bilayer observed with related systems. The interaction is stronger for cationic than anionic surfaces, which is rationalized in terms of the negative zeta potential of the LCNPs. The work shows that the attachment of and spreading from LCNPs is influenced by the properties of the surface, the internal structure, composition and stability of the particles as well as the nature of the stabilizer.

Contribution of Antibody Hydrodynamic Size to Vitreal Clearance Revealed through Rabbit Studies Using a Species-Matched Fab.

We have developed a tool Fab fragment of a rabbit monoclonal antibody that is useful for early evaluation in rabbit models of technologies for long acting delivery (LAD) of proteins to the eye. Using this Fab we show that vitreal clearance can be slowed through increased hydrodynamic size. Fab (G10rabFab) and Fab' (G10rabFab') fragments of a rabbit monoclonal antibody (G10rabIgG) were expressed in Chinese hamster ovary (CHO) cells and purified using antigen-based affinity chromatography. G10rabFab retains antigen-binding upon thermal stress (37 °C) for 8 weeks in phosphate-buffered saline (PBS) and can be detected in rabbit tissues using an antigen-based ELISA. Hydrodynamic radius, measured using quasi-elastic light scattering (QELS), was increased through site-specific modification of the G10rabFab' free cysteine with linear methoxy-polyethylene glycol(PEG)-maleimide of 20000 or 40000 molecular weight. Pharmacokinetic studies upon intravitreal dosing in New Zealand white rabbits were conducted on the G10rabFab and PEGylated G10rabFab'. Results of single and multidose pharmacokinetic experiments yield reproducible results and a vitreal half-life for G10rabFab of 3.2 days. Clearance from the eye is slowed through increased hydrodynamic size, with vitreal half-life showing a linear dependence on hydrodynamic radius (RH). A linear dependence of vitreal half-life on RH suggests that molecule diffusivity makes an important contribution to vitreal clearance. A method for prediction of vitreal half-life from RH measurements is proposed.

Investigation of Fragment Antibody Stability and Its Release Mechanism from Poly(Lactide-co-Glycolide)-Triacetin Depots for Sustained-Release Applications.

Achieving long-term drug release from polymer-based delivery systems continues to be a challenge particularly for the delivery of large hydrophilic molecules such as therapeutic antibodies and proteins. Here, we report on the utility of an in situ-forming and injectable polymer-solvent system for the long-term release of a model antibody fragment (Fab1). The delivery system was prepared by dispersing a spray-dried powder of Fab1 within poly(lactide-co-glycolide) (PLGA)-triacetin solution. The formulation viscosity was within the range 1.0 ± 0.3 Pa s but it was injectable through a 27G needle. The release profile of Fab1, measured in phosphate-buffered saline (PBS), showed a lag phase followed by sustained-release phase for close to 80 days. Antibody degradation during its residence within the depot was comparable to its degradation upon long-term incubation in PBS. On the basis of temporal changes in surface morphology, stiffness, and depot mass, a mechanism to account for the drug release profile has been proposed. The unprecedented release profile and retention of greater than 80% of antigen-binding capacity even after several weeks demonstrates that PLGA-triacetin solution could be a promising system for the long-term delivery of biologics.

Non-lamellar lipid liquid crystalline structures at interfaces.

The self-assembly of lipids leads to the formation of a rich variety of nano-structures, not only restricted to lipid bilayers, but also encompassing non-lamellar liquid crystalline structures, such as cubic, hexagonal, and sponge phases. These non-lamellar phases have been increasingly recognized as important for living systems, both in terms of providing compartmentalization and as regulators of biological activity. Consequently, they are of great interest for their potential as delivery systems in pharmaceutical, food and cosmetic applications. The compartmentalizing nature of these phases features mono- or bicontinuous networks of both hydrophilic and hydrophobic domains. To utilize these non-lamellar liquid crystalline structures in biomedical devices for analyses and drug delivery, it is crucial to understand how they interact with and respond to different types of interfaces. Such non-lamellar interfacial layers can be used to entrap functional biomolecules that respond to lipid curvature as well as the confinement. It is also important to understand the structural changes of deposited lipid in relation to the corresponding bulk dispersions. They can be controlled by changing the lipid composition or by introducing components that can alter the curvature or by deposition on nano-structured surface, e.g. vertical nano-wire arrays. Progress in the area of liquid crystalline lipid based nanoparticles opens up new possibilities for the preparation of well-defined surface films with well-defined nano-structures. This review will focus on recent progress in the formation of non-lamellar dispersions and their interfacial properties at the solid/liquid and biologically relevant interfaces.

Interactions of PAMAM dendrimers with negatively charged model biomembranes.

We have investigated the interactions between cationic poly(amidoamine) (PAMAM) dendrimers of generation 4 (G4), a potential gene transfection vector, with net-anionic model biomembranes composed of different ratios of zwitterionic phosphocholine (PC) and anionic phospho-L-serine (PS) phospholipids. Two types of model membranes were used: solid-supported bilayers, prepared with lipids carrying palmitoyl-oleoyl (PO) and diphytanoyl (DPh) acyl chains, and free-standing bilayers, formed at the interface between two aqueous droplets in oil (droplet interface bilayers, DIBs) using the DPh-based lipids. G4 dendrimers were found to translocate through POPC:POPS bilayers deposited on silica surfaces. The charge density of the bilayer affects translocation, which is reduced when the ionic strength increases. This shows that the dendrimer-bilayer interactions are largely controlled by their electrostatic attraction. The structure of the solid-supported bilayers remains intact upon translocation of the dendrimer. However, the amount of lipids in the bilayer decreases and dendrimer/lipid aggregates are formed in bulk solution, which can be deposited on the interfacial layers upon dilution of the system with dendrimer-free solvent. Electrophysiology measurements on DIBs confirm that G4 dendrimers cross the lipid membranes containing PS, which then become more permeable to ions. The obtained results have implications for PAMAM dendrimers as delivery vehicles to cells.