Browsing by Author "Kastellorizios, Michail"
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Item 3D-Printed Insert for Visualization of Liposomal Interactions with Collagen Fibers(2019-03-05) Mishra, Ina; Kastellorizios, Michail; Curry, StephenPurpose: Nanoparticles, such as liposome, are commonly used as vehicles for drug delivery to target tissues such as tumors. However, relatively little research has focused on liposomal interactions with the tumor extracellular matrix (ECM), a huge hurdle in the process of perfecting dosage. To our knowledge, there is no method available that allows the observation liposomes against a whole collagen fiber. In order to better study these interactions, we have designed a 3D-printed frame that helps in visualizing liposome transport around a collagen fiber. Observation of our replicated microenvironment will allow us to shed more light on the movements of liposomes within the ECM of tumor cells. Methods: Collagen fiber was removed from adult rat tail and allowed to soak in phosphate-buffered saline (PBS) overnight. Primary design of the frame was done using SketchUp software. Printing of 12mm frame design was done using a Form 2 3D printer (Formlabs). Fiber-in-frame was placed in a glass bottom microwell dish (MatTek Corp.). Research-grade liposomes that mimic the clinical product Doxil (DoxomTM, Liposomics) were used in a background of 0.07mg/ml BSA solution, and added to the channel. Visualization of the interaction was done using confocal microscope (Nikon A1R-MP+ Multiphoton System). Results: As a proof-of-concept, we mounted a collagen fiber into our frame and filled channel with BSA solution. Observation indicates that liposomes prefer to accumulate on the collagen fiber surface. Multiple trial frames were needed to account for height requirements necessary for confocal microscopy. Our built-in sample well allowed for a maximum 20 uL of formulation to be added into the channel environment from one end of the fiber. Our next steps will focus on reducing height and volume of background and sample needed as well as removing the need for a dish or slide altogether. Future studies will focus on kinetic phenomena in this microenvironment. Conclusions: Using the methods described above, we observed that Doxom liposomes accumulated near the fiber surface. This suggests that liposomes can use collagen as a route of travel in the extracellular environment. Future experiments with this frame design and others will be used to study the liposomal interactions with collagen and other ECM components. In addition to new frame designs, instruments with higher intensity and resolution will provide more accurate data.Item A Novel Method to Characterize Invasive Ductal Carcinoma Tumor Biopsies Using Contact Angle Measurements(2022) Rincon, Julio; Mishra, Ina; Kastellorizios, MichailPurpose: The goal of this work is to develop a preclinical method to characterize human breast cancer biopsies of different racial origin. Contact angle measurements are used to assess the biopsies' surface properties and examine possible correlation with race/ethnicity, tumor type, and cancer grade. This method enables us to study differences in interaction of drugs directly in tumor tissues based on available covariate data of the obtained samples. Here, we present a study of 80 invasive ductal carcinoma tumor samples compared against their matching normal and/or cancer adjacent tissues. Methods: To obtain contact angles of tumor tissues, we developed a contact angle instrument capable of delivering a ~45 nL drop on top of a 1.5 mm biopsy using a modified goniometer with added custom components (DataPhysics Instruments USA Corp.). The system allows us to measure contact angles from 3 different positions (0°, -45°, 90°), and viewing the tumor tissue through an inverted microscope to determine drop position and quality. For this study, breast cancer tissues were obtained as tumor micro arrays (TMA) and as FFPE tissues with matching normal adjacent tissue. For TMA slides, two drops per tissue were delivered and the test was repeated with a subsequent section of the same TMA, unless the TMA included duplicate cores. For FFPE samples, tissues were processed with a microtome at a thickness of 15 µm, a minimum of 6 drops were delivered per tissue. Results: Aggregated data showed normal adjacent tissue (NAT) had an average contact angle (CA) of 51.4° ± 6.5° n=19, cancer adjacent tissue (CAT) had an average CA of 62.8° ± 8° n=59, grade 1 tumors had an average CA of 71.1° ± 7° n=13, grade 2 tumors had an average CA of 67.4 ± 9.1° n=49, and grade 3 tumors had an average CA of 64.6° ± 10.3° n=11. When comparing normal adjacent tissue against any other tissue, p-values ≤ 0.05 and power ≥ 0.80 were observed. When comparing cancer adjacent tissue, only CAT vs NAT and CAT vs grade 2 tumors had p-values ≤ 0.05 and power ≥ 0.80. Individually, 37 cases reached p-values ≤ 0.05 and power ≥ 0.80, were tumor tissue showed contact angles greater than their NAT or CAT. An additional 7 cases met p-values ≤ 0.05 and power ≥ 0.80, however, tumor contact angles were lower than their NAT or CAT. Finally, ignoring type 2 errors, then an additional 11 cases reached p-values ≤ 0.05. Conclusions: Higher contact angles of deionized water were observed in tumor tissues when compared to matching normal or cancer adjacent tissue. It is clear that breast cancer tumors exhibit surface energy differences from normal adjacent tissues, with cancer tissue being more hydrophobic compared to normal tissue. Future work includes the determination of contact angles of Doxil-like liposomes in these tumors and the determination of surface energy of the tissues.Item An Alternative Method To Quantify Surface Properties of Anti-Cancer Drugs(2019-03-05) Curry, Stephen; Kastellorizios, Michail; Mishra, InaPurpose Physicochemical properties such as size and size distribution affect liposome formulations’ physical stability and accumulation in the target tissue. The FDA’s “Liposome Drug Products, Guidance for Industry”, 2018 emphasized size and size-distribution as “critical quality attributes”, however, it does not mention the criteria for an acceptable polydispersity index (PDI), currently measured using size-scattering technique. A monodisperse, homogenous size distribution population (PDI≤0.3) is desired. In this work, we measured surface tension of two different size distributions of liposome populations, with lipid composition similar to clinically approved anticancer formulation DOXIL, as a method to quantify liposome surface properties. This work establishes a building block in our long-term goal of obtaining insight into and facilitating the translation of nanoparticles from animal to human studies by offering additional preclinical characterization techniques based on surface properties of nanoparticles. Method Liposome formulations A and B, consisting of hydrogenated (Soy) phosphatidyl choline, cholesterol, and DSPE-PEG (7.64:5.17:1 molar ratio) were formulated in-house using thin film hydration method and probe sonication. Average particle size (PS) and PDI was obtained using dynamic light scattering (Mobius122, Wyatt Technology). The two formulations were fabricated with small differences in probe sonication process, yielding slightly different PDIs but the same average PS. The formulations were tested at two different lipid concentrations: 1mg/ml and 0.1mg/ml. Surface energy measurements were obtained on all four using pendant drop method, reported as mean with standard error (n=3) against the PDI. Results Average PS for A and B was similar (92.9±1.6 nm, 90.3±0.4 nm respectively), with PDIs 0.1±0.02 and 0.04±0.02 respectively. As expected, the surface tension was significantly decreased with concentration. PDI was found to significantly affect the total surface tension at higher concentration tested (1mg/ml) while it did not play a role at the lower concentration tested (0.1mg/ml). Interestingly, this trend was reversed when the surface tension was broken into its polar and dispersive components. Conclusions This work confirmed that small PDI changes, arising from slight variations in fabrication/manufacturing process, can translate into measurable changes in surface properties that can be obtained more rapidly and with higher accuracy than conventional DLS-based sizing techniques.Item Characterization for In-situ Ocular Implant Formation(2024-03-21) Khuu, Megan; Kastellorizios, MichailPurpose Different diseases of the eye require intravitreal injections for treatment. While intravitreal injections are quick procedures, their requirement for routine in-office visits makes them inconvenient. An alternative is intra-ocular implants, designed to stay within the eye, these allow controlled release of therapeutic drugs to the posterior segment of the eye. Intra-ocular implants require symmetrical shapes for even and steady release of the drug. To study how well in-situ implants form, vitreous humor substitutes were made using varying ratios of hyaluronic acid (HA) and polyvinyl alcohol (PVA) within phosphate-buffered saline (PBS). These substitutes have surface characteristics that can be used as a potential marker to predict how symmetrical the in-situ implant forms. Here, we use contact angles to help characterize their interaction. Methods Vitreous humor substitutes were made using 0.01 g/mL solutions of HA and PVA in PBS. These solutions were mixed to create the following ratios of HA:PVA: 1:3, 1:1, and 3:1. To make the implant, 1-Methyl-2-pyrrolidone was dissolved in polylactic-co-glycolic acid in a 1.96:1 ratio. Films were prepared on glass slides using the solutions to take contact angles of the implant. Contact angles were taken using an optical goniometer at 30.2 frames per second for 10 seconds, where it was set to dispense 2 µL each time. For in-situ implant formation, the implant was injected into 5 mL wells of each solution (PBS, HA, PVA, 1:3, 1:1, and 3:1) at a 90-degree angle using a 20 gauge needle at 1.2 cm deep. In-situ implant formation was repeated 4 times. Results The following contact angles were taken at frame 300 and are the average of at least 10 runs. The average contact angle for water on a plain glass slide and the implant on a plain glass slide, PVA film, 1:3 film, 1:1 film, 3:1 film, and on the HA film was 26.13 ± 3.79, 28.57 ± 2.29, 32.65 ± 5.75, 32.41 ± 4.21, 35.02 ± 3.03, 37.20 ± 4.92, and 40.71 ± 5.22 degrees respectively. In-situ implant formation was consistently the most symmetrical within HA and the 1:3 hybrid, though still in irregular yet compact shapes. In-situ implant formations within the other solutions were randomly shaped with tendrilous protrusions that would coil upon themselves. Conclusions Contact angles for all solutions were found to be statistically different using T-tests. The different contact angles allow us to manipulate the interactions by varying the ratio of HA and PVA to make an environment most suitable for intra-ocular implant formation. Interestingly, the 1:3 substitute did not follow the direct relationship found between the amount of HA present in each vitreous humor substitute and their contact angles. Despite having one of the least amounts of HA, it provided a better environment for implant formulation. Thus, there must be an interaction between HA, PVA, and the implant that is optimal at 1:3. More is to be done with the 1:3 hybrid and implant by repeating the experiment with serial dilutions to help us determine which patient populations may be most suitable to this type of treatment.Item Effect of Cholesterol Content on Surface Properties of Doxil-Mimicking Liposomes(2019-03-05) Garrett, Meredith; Mishra, Ina; Kastellorizios, Michail; Jameson, JeffreyPurpose:The use of nanoparticles (NPs) for drug delivery has gained a lot of attention from biomedical researchers in the last few decades. This is primarily because NPs are of an extremely small size that enables them to accumulate preferentially inside of the tumors instead of healthy tissue. The reason is unknown as to why research being done on NPs has shown promising results in animal models but does not translate successfully to the clinic. Here, we will study the effect of NPs cholesterol content on their surface tension. Cholesterol is sometimes added to NPs to increase their rigidity and stability, but we do not know if it interferes with other properties such as surface tension. We hypothesize that surface tension can be useful in enriching characterization of novel NPs and help determine which NPs to move on to clinical testing. Methods:We used Doxil®, one of the few successful NPs for cancer therapy, as reference. Doxil® consists of a drug (doxorubicin) encapsulated in nano-sized vehicles (liposomes) made of HSPC, cholesterol (CHOL), and PEG. Two formulations of NPs, HSPC:CHOL:PEG (Doxil®) and HSPC:PEG, were prepared by thin film hydration followed by membrane extrusion. We tested their surface tension at six concentrations using the twin-capillary method. A capillary stand was 3D-printed that was able to hold the capillaries stable. Results: The average change in surface tension for the six different concentrations between the two formulations did not have a noticeable change. The surface tension decreased proportionately at the same concentrations for each of the HSPC:CHOL:PEG and HSPC:PEG formulations. In addition, the same experiment was used to compare the difference in surface tension between HSPC:CHOL and phosphatidylcholine (PC), which is equivalent to HSPC. The difference between the values were also negligible. Conclusion:Based on our results, we conclude that the inclusion of cholesterol in liposomes does not alter their surface energy properties as measured by the twin capillary rise method. This may be due to the fact that cholesterol is a small molecule while PEG is a giant molecule that covers the surface of the entire liposome. Even though cholesterol does not affect surface tension, it is still pertinent for the formulation to be effective. In future studies, an additional technique will be used (pendant drop shape analysis) that gives more insight into surface tension and breaks it into its polar and non-polar components.Item Effect of Composition and Size on Surface Properties of Anti-Cancer Nanoparticles(MDPI, 2023-09-09) Mishra, Ina; Garrett, Meredith; Curry, Stephen; Jameson, Jeffrey; Kastellorizios, MichailLiposomal formulations offer significant advantages as anticancer drug carriers for targeted drug delivery; however, due to their complexity, clinical translation has been challenging. In addition, liposomal product manufacturing has been interrupted in the past, as was the case for Doxil((R)) (doxorubicin hydrochloride liposome injection). Here, interfacial tension (IFT) measurements were investigated as a potential physicochemical characterization tool to aid in liposomal product characterization during development and manufacturing. A pendant drop method using an optical tensiometer was used to measure the interfacial tension of various analogues of Doxil((R)) liposomal suspensions in air and in dodecane. The effect of liposome concentration, formulation (PEG and cholesterol content), presence of encapsulated drug, as well as average particle size was analyzed. It was observed that Doxil((R)) analog liposomes demonstrate surfactant-like behavior with a sigmoidal-shape interfacial tension vs. concentration curve. This behavior was heavily dependent on PEG content, with a complete loss of surfactant-like behavior when PEG was removed from the formulation. In addition to interfacial tension, three data analyses were identified as able to distinguish between formulations with variations in PEG, cholesterol, and particle size: (i) polar and non-polar contribution to interfacial tension, (ii) liposomal concentration at which the polar and non-polar components were equal, and (iii) rate of interfacial tension decay after droplet formation, which is indicative of how quickly liposomes migrate from the bulk of the solution to the surface. We demonstrate for the first time that interfacial tension can be used to detect certain liposomal formulation changes, such as PEG content, encapsulated drug presence, and size variability, and may make a useful addition to physicochemical characterization during development and manufacturing of liposomal products.Item Effect of increased blood glucose on anti-cancer nanoparticle interactions with tissue(2020) Kastellorizios, Michail; Mishra, Ina; Nguyen, AlexandraPurpose: Little research has been conducted on liposomal drug delivery in pediatric diabetics and non-diabetics with cancer. Preliminary data demonstrates that increased liposomal interactions with collagen in the tumor's extracellular matrix affect tumor targeting. The goal of the present study was to understand the effect that glucose has on liposomes' interaction with collagen and its interference with the anti-cancer action of liposomal drugs in diabetic pediatric patients. Methods: Liposomes were formulated according to the Doxil molar ratio. Varying amounts of glucose, glycated albumin (GA), albumin, and liposomes were dissolved into phosphate-buffered saline (PBS) to make solutions representative of the mean pediatric hypoglycemic, normoglycemic, and hyperglycemic diabetic and non-diabetic patient. An Optical Contact Angle instrument quantified the interactions between liposomes and collagen by advancing and receding a drop of each solution onto human and rat collagen-coated slides. Results: The contact angle measured was lower in human collagen than rat collagen in the PBS and Liposomes samples. In samples with added glucose and albumin, the contact angle was lower in rat collagen than human collagen. Conclusions: Liposomes by themselves in PBS have a greater interaction with human collagen than rat collagen, but the addition of glucose, GA, and albumin reverses this trend so that liposomes have a greater interaction with rat collagen than human collagen. The protein's interactions also overpower the liposomes' interaction with collagen. Further investigation is necessary to determine if the presence of elevated glucose has a significant effect on how liposomes interact with collagen.Item Investigating Effects of Pegylation of the Surface Tension of Liposomes used in the Treatment of Breast Cancer(2019-03-05) Jameson, Jeffrey; Mishra, Ina; Kastellorizios, Michail; Garrett, MeredithPurpose: In recent years there have been many advancements in nanomedicine as potential delivery systems for the treatment of various types of cancer. Two formulations on the market, Doxil® and Myocet®, are made of doxorubicin-loaded liposomes. One them, Doxil® has the hydrophilic polymer polyethylene glycol (PEG) immobilized on its surface, while the other (Myocet®) doesn’t. PEG has been shown to improve liposome circulation in the blood by disguising them from the immune system, creating a ‘stealth’ delivery system for drugs. However, it is unknown whether the PEG changes the way liposomes may interact within the tumor. Here, we fabricated liposomes that mimic Doxil® and Myocet® and measured their surface tension, as surface tension is a driving force behind the interfacial interactions between liposomes and tumor extracellular matrix. Materials and Methods: Two formulations of liposomes (Doxil® and Myocet®) were fabricated using thin film hydration for liposome formation and membrane extrusion for liposome downsizing to nanometer levels. A thin film of each formulation was made and then hydrated with phosphate buffered saline. Extrusion was performed using a 50nm membrane bringing final liposome size of each formulation to approximately 100nm. The Doxil® formulation contained hydrogenated soybean phosphatidylcholine (HSPC), cholesterol and PEG, while the Myocet® contained only HSPC and cholesterol. The two formulations underwent a series of dilutions, and the surface tension of each concentration was calculated using the twin capillary rise method. A custom 3D printed stand coupled with a well plate was used to secure the capillaries while allowing small volumes of the formulations to be used. Results: Data collected showed a significant decrease in surface tension when PEG is removed from the surface of the liposomes. This effect was consistent across all concentrations when compared to the original formulation. Conclusions: Our results show that PEG causes a drastic change in how liposomes interact with surfaces. Further, we demonstrated that this effect is greatly affected by small changes in liposome concentration. The reduction in surface tension observed in Myocet® versus Doxil® may be the reason behind Doxil’s sustained success in the clinic compared to Myocet®. Future studies will focus on quantifying interfacial tension between these liposome formulations and breast cancer biopsies in order to potentially explain their different clinical performance.Item Novel Method to Obtain Contact Angles of Tumor Biopsies(American Chemical Society, 2023-08-07) Rincon, Julio; Kastellorizios, MichailCharacterizing the strength of a solid-liquid interface can be done by depositing a single drop of liquid on a planar solid surface and measuring the angle of the formed semicircle, called the contact angle. The contact angle of pure water is indicative of a surface's hydrophobicity and is a useful metric in biomedical applications such as tissue scaffolding and drug/tissue interactions. However, the roughness and inhomogeneity of most biological surfaces make obtaining accurate contact angles of such materials challenging. Here, we developed an instrument and methodology to obtain contact angles of tissue sections. Breast cancer tumor and nearby healthy tissue sections were used as the model biological surface. The custom instrument was built on existing equipment by improving drop dispensing accuracy in the nanoliter range, an XYZ stage, additional side view cameras, and microscope-based sample visualization. The method takes into account the inherent surface inhomogeneity and topology of tissue and the required method of illumination for contact angle acquisition. As such, the system uses an inverted microscope with a high sensitivity camera, an XYZ stage for accurate droplet placement on tissue, and multiple cameras to obtain contact angles around the entire perimeter of the drop. We tested the system with breast cancer biopsies and adjacent normal tissue from 75 patients and report here a trend of tumor exhibiting higher water contact angles, and thus higher hydrophobicity, compared to their respective normal adjacent tissue. The system described here can be used to characterize any type of biological tissue, which can be sectioned, with any liquid including water or solutions with dissolved or suspended therapeutic molecules and particles.Item Pharmacokinetic and Physicochemical Evaluation of Novel Drug Candidates for Retinitis Pigmentosa(2023) Garrett, Meredith; Curry, Stacy; Feris, Sherri; Martin, Stephen; Clark, Abbot; Kastellorizios, MichailPurpose: Retinitis pigmentosa is a set of inherited ocular diseases that affect nearly 3 million people worldwide. The condition is inherited and causes the progressive deterioration of the retina. Retinitis pigmentosa begins with the loss of rod photoreceptors which cause night blindness and a decrease in peripheral vision. After significant loss of rod cells, cone cells also begin to die, decreasing central vision until complete blindness. More than 150 genetic mutations in 80 different genes have thus far been identified to contribute to progression pathways of the condition. Despite ongoing stem cell and gene therapy investigations, thus far there are no curative options. Most existing treatments focus on slowing the progression of retinal deterioration by reducing oxidative stress on the retina. Unfortunately, these treatments only achieve limited success and cannot halt progression. Recently, the sigma 2 receptor (σ2r) was identified to be endoplasmic reticulum membrane protein 97 (TMEM97). This protein (σ2r/TMEM97) has been shown to have neuroprotective effects on retinal cells and is thus of interest as a potential drug target for retinitis pigmentosa. Here we synthesized and tested a series of six compounds which have previously been found to modulate σ2r/TMEM97. To determine which of these compounds is a suitable drug candidate, each underwent in vivo and in vitro testing with the goal of selecting the best candidate for further clinical development. Methods: We tested the compounds in a rat model to determine retinal uptake following intravitreal injection. Each drug was dissolved in dimethyl sulfoxide (DMSO) and injected into the eye. At set time points, animals were sacrificed, and retinas were isolated from harvested eyes. The retina was separated and homogenized using sonication. A small portion was removed and underwent protein precipitation to purify the sample. The samples were then analyzed via liquid chromatography mass spectrometry (LCMS) to find the drug concentration remaining at each timepoint. In addition to obtaining a pharmacokinetic profile, the compounds were physiochemically characterized for chemical stability, solubility, in vitro drug release from vitreous humor, thermal analysis, and surface tension. Conclusion: Our goal is to select those drug candidates with the highest chance of clinical success. The pharmacokinetic profiles as well as physicochemical characteristics and stability of the compounds obtained in this study revealed important differences between the compounds that were used in selecting which to advance to in vivo efficacy testing. Ongoing studies include completion of physicochemical characterization and in vivo efficacy in a retinitis pigmentosa rat model that will be used to identify top candidates for further development.Item Preclinical Characterization of Novel Drug Candidates for Ocular Drug Delivery(2024-03-21) Garrett, Meredith; Kastellorizios, MichailPurpose: Retinitis pigmentosa (RP) is the leading cause of vision loss and blindness for people under 60 years old. RP is an inherited disease causing progressive and irreversible deterioration of the retina. To date, over 150 mutations in 90 genes have been identified to contribute to the disease through various pathways. Except for a single mutation responsible for less than 5% of cases, RP is incurable. Currently available treatments largely focus on slowing progression by relieving oxidative stress and are met with limited success. The sigma 2 receptor, also established as transmembrane protein 97 of the endoplasmic reticulum (s2r/TMEM97), has been shown to have neuroprotective effects on retinal cells and is a potential drug target for RP. Recently, a series of novel drug compounds have been identified to modulate the s2r/TMEM97 protein and are under investigation as possible candidates for treatment of RP. Here, as part of preclinical evaluation, we performed thermal analysis of the novel compounds, including thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). These techniques give insight to drug behavior at different temperatures and provide information on stability and structure. We also conducted a series of drug release studies which monitored movement of the compounds from vitreous humor through a dialysis membrane. This will allow for correlation with previously collected data. Methods: TGA was performed on a TGA550 thermogravimetric analyzer from TA Instruments. The instrument measured the mass of the pan as the temperature was increased from 20°C to 700°C at a rate of 10°C/min. DSC was completed using a DSC250 from TA Instruments. The instrument was programed to increase the temperature of the sample chamber from 10°C to 200°C at a rate of 5°C/min while measuring the heat flux of each pan. A series of drug release studies were performed using a specialized dialysis plate. Drug movement from vitreous humor across a membrane was evaluated over a 12-hour period. Results: The data collected here for the novel compounds did not show any red flags which would indicate a poor drug candidate. TGA data showed all compounds were thermally stabile until approximately 175°C, at which point they began to lose mass. DSC differential thermograms did not exhibit crystalline behavior. Drug release studies did not show a strong interaction between the compounds and vitreous humor. Conclusion: Our goal is to aid in narrowing the series of novel drug compounds by providing robust preclinical characterization. TGA thermograms obtained demonstrated the compounds were thermally stabile up to approximately 175°C, which is standard for small molecules. DSC results reveal the compounds are not crystalline and indicate the need for a special formulation. The drug release studies show there were no strong interactions with the compounds and vitreous humor. The data here was included with stability, solubility, and in vitro and in vivo pharmacokinetic analysis and used to select the two leading candidates to advance to in vivo efficacy studies in a transgenic rat model for retinitis pigmentosa.Item Real-time monitoring of membrane composition for liposomal drug formulations in continuous manufacturing.(2021) Rincon, Julio; Garrett, Meredith; Kastellorizios, MichailPurpose: Liposomes are lipid-based vesicles with the ability to entrap drugs. Their applications continue to benefit from advances in technology and manufacturing. When it comes to liposomes, or any nanomedicines, attributes such as membrane composition, size, and drug encapsulation are important to consistently deliver the intended performance of drug products. Newly adopted continuous manufacturing processes present challenges for real-time critical attribute analysis, as most characterization techniques employ a batch-based process. Here, we present a methodology to verify liposome membrane composition in a manner that can be implemented in continuous manufacturing process by detecting changes in surface tension. Methods: 8 capillary tubes (25 µl) were simultaneously submerged in 96 well plates. Wells were loaded with 250 µl of a Doxil-like liposome formulation at a concentration of 16 mg/ml. DSPEPEG ratios varied from 2.6%, 3.9%, 4.5%, 5.3%, 6%, 8%, 9%. Cholesterol molar ratio was kept at 38%, HSPC content varied dependent on DSPEPEG ratio. Three different protocols were evaluated: single 10-second submersion, 45 continuous submersions (500 milliseconds each), and 25 µl prefill followed by 15 submersions. Results: Rise measurements demonstrated significant surface tension differences between 5.3% and all other ratios using the prefill method. In addition, both single and multiple immersion protocols showed capillary rise hysteresis. Conclusions: Preliminary studies verify surface tension can be used to distinguish PEG variations in liposome membrane composition. Additional protocol and method development is required to reduce capillary rise hysteresis and further enable real-time monitoring of membrane composition in continuous manufacturing.