Membrane-Targeting Perylenylethynylphenols Inactivate Medically Important Coronaviruses via the Singlet Oxygen Photogeneration Mechanism.

Perylenylethynyl derivatives have been recognized as broad-spectrum antivirals that target the lipid envelope of enveloped viruses. In this study, we present novel perylenylethynylphenols that exhibit nanomolar or submicromolar antiviral activity against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) and feline infectious peritonitis virus (FIPV) in vitro. Perylenylethynylphenols incorporate into viral and cellular membranes and block the entry of the virus into the host cell. Furthermore, these compounds demonstrate an ability to generate singlet oxygen when exposed to visible light. The rate of singlet oxygen production is positively correlated with antiviral activity, confirming that the inhibition of fusion is primarily due to singlet-oxygen-induced damage to the viral envelope. The unique combination of a shape that affords affinity to the lipid bilayer and the capacity to generate singlet oxygen makes perylenylethynylphenols highly effective scaffolds against enveloped viruses. The anticoronaviral activity of perylenylethynylphenols is strictly light-dependent and disappears in the absence of daylight (under red light). Moreover, these compounds exhibit negligible cytotoxicity, highlighting their significant potential for further exploration of the precise antiviral mechanism and the broader scope and limitations of this compound class.

Use of droplet-based microfluidic techniques in the preparation of microparticles.

Microparticles are widely used in myriad fields such as pharmaceuticals, foods, cosmetics, and other industrial fields. Compared with traditional methods for synthesizing microparticles, microfluidic techniques provide very powerful platforms for creating highly controllable emulsion droplets as templates for fabricating uniform microparticles with advanced structures and functions. Microfluidic techniques can generate emulsion droplets with precisely controlled size, shape, and composition. A more precise preparation process brings an effective tool to control the release profile of the drug and introduces an easily accessible reproducibility. The paper gives information about basic droplet-based set-ups and examples of attainable microparticle types preparable by this method.

Use of droplet-based microfluidic techniques in the preparation of microparticles.

Microparticles are widely used in myriad fields such as pharmaceuticals, foods, cosmetics, and other industrial fields. Compared with traditional methods for synthesizing microparticles, microfluidic techniques provide very powerful platforms for creating highly controllable emulsion droplets as templates for fabricating uniform microparticles with advanced structures and functions. Microfluidic techniques can generate emulsion droplets with precisely controlled size, shape, and composition. A more precise preparation process brings an effective tool to control the release profile of the drug and introduces an easily accessible reproducibility. The paper gives information about basic droplet-based set-ups and examples of attainable microparticle types preparable by this method.

Preformulation design of PLGA particulate system for multi-day drug delivery of the antidepressant mirtazapine.

In this experimental study, the biodegradable polylactide-co-glycolide (PLGA) microparticles (MP) loaded with the insoluble antidepressant mirtazapine were prepared by the simple o/w solvent evaporation method. The formation involved intrinsic variables, such as the content of polymer (700, 900 or 1200 mg), dichloromethane (5 or 10 ml) and/or drug (200 or 400 or 600 mg), and the volume of the aqueous emulsion phase (400, 600 or 800 ml). The influence of these parameters on the size and morphology of microparticles, encapsulation efficiency, and drug release behavior was observed. All MP were successfully prepared, and their size ranged between 165.34 ± 42.88 and 360.17 ± 121.59 μm. MP exhibited prolonged drug release (days), and some profiles had multiphasic character. It was found that the samples prepared with a  higher initial amount of PLGA were bigger with prolonged lag time up to 34.3 hours. On the other hand, higher drug concentrations reduced the lag time. The external phase volume reduction and multiplication of dichloromethane amount prolonged the mirtazapine release and decreased the encapsulation efficiency. These observations were further confirmed by multivariate data analysis.

Application of Advanced Microscopic Methods to Study the Interaction of Carboxylated Fluorescent Nanodiamonds with Membrane Structures in THP-1 Cells: Activation of Inflammasome NLRP3 as the Result of Lysosome Destabilization.

Nanodiamonds (ND), especially fluorescent NDs, represent potentially applicable drug and probe carriers for in vitro/in vivo applications. The main purpose of this study was to relate physical-chemical properties of carboxylated NDs to their intracellular distribution and impact on membranes and cell immunity-activation of inflammasome in the in vitro THP-1 cell line model. Dynamic light scattering, nanoparticle tracking analysis, and microscopic methods were used to characterize ND particles and their intracellular distribution. Fluorescent NDs penetrated the cell membranes by both macropinocytosis and mechanical cutting through cell membranes. We proved accumulation of fluorescent NDs in lysosomes. In this case, lysosomes were destabilized and cathepsin B was released into the cytoplasm and triggered pathways leading to activation of inflammasome NLRP3, as detected in THP-1 cells. Activation of inflammasome by NDs represents an important event that could underlie the described toxicological effects in vivo induced by NDs. According to our knowledge, this is the first in vitro study demonstrating direct activation of inflammasome by NDs. These findings are important for understanding the mechanism(s) of action of ND complexes and explain the ambiguity of the existing toxicological data.

Hyaluronic Acid Surface Modified Liposomes Prepared via Orthogonal Aminoxy Coupling: Synthesis of Nontoxic Aminoxylipids Based on Symmetrically α-Branched Fatty Acids, Preparation of Liposomes by Microfluidic Mixing, and Targeting to Cancer Cells Expressing CD44.

New synthetic aminoxy lipids are designed and synthesized as building blocks for the formulation of functionalized nanoliposomes by microfluidization using a NanoAssemblr. Orthogonal binding of hyaluronic acid onto the outer surface of functionalized nanoliposomes via aminoxy coupling ( N-oxy ligation) is achieved at hemiacetal function of hyaluronic acid and the structure of hyaluronic acid-liposomes is visualized by transmission electron microscopy and cryotransmission electron microscopy. Observed structures are in a good correlation with data obtained by dynamic light scattering (size and ζ-potential). In vitro experiments on cell lines expressing CD44 receptors demonstrate selective internalization of fluorochrome-labeled hyaluronic acid-liposomes, while cells with down regulated CD44 receptor levels exhibit very low internalization of hyaluronic acid-liposomes. A method based on microfluidization mixing was developed for preparation of monodispersive unilamellar liposomes containing aminoxy lipids and orthogonal binding of hyaluronic acid onto the liposomal surface was demonstrated. These hyaluronic acid-liposomes represent a potentially new drug delivery platform for CD44-targeted anticancer drugs as well as for immunotherapeutics and vaccines.

Electrochemical Platform for the Detection of Transmembrane Proteins Reconstituted into Liposomes.

The development of new methods and strategies for the investigation of membrane proteins is limited by poor solubility of these proteins in an aqueous environment and hindered by a number of other problems linked to the instability of the proteins outside lipid bilayers. Therefore, current research focuses on an analysis of membrane proteins incorporated into model lipid membrane, most frequently liposomes. In this work, we introduce a new electrochemical methodology for the analysis of transmembrane proteins reconstituted into a liposomal system. The proposed analytical approach is based on proteoliposomal sample adsorption on the surface of working electrodes followed by analysis of the anodic and cathodic signals of the reconstituted proteins. It works based on the fact that proteins are electroactive species, in contrast to the lipid components of the membranes under the given experimental conditions. Electroanalytical experiments were performed with two transmembrane proteins; the Na(+)/K(+)ATPase that contains transmembrane as well as large extramembraneous segments and the mitochondrial uncoupling protein 1, which is a transmembrane protein essentially lacking extramembraneous segments. Electrochemical analyses of proteoliposomes were compared with analyses of both proteins solubilized with detergents (C12E8 and octyl-PoE) and supported by the following complementary methods: microscopy techniques, protein activity testing, molecular model visualizations, and immunochemical identification of both proteins. The label-free electrochemical platform presented here enables studies of reconstituted transmembrane proteins at the nanomolar level. Our results may contribute to the development of new electrochemical sensors and microarray systems applicable within the field of poorly water-soluble proteins.

Molecular adjuvants based on nonpyrogenic lipophilic derivatives of norAbuMDP/GMDP formulated in nanoliposomes: stimulation of innate and adaptive immunity.

PURPOSE: The aim of this work was to demonstrate an immunostimulatory and adjuvant effect of new apyrogenic lipophilic derivatives of norAbuMDP and norAbuGMDP formulated in nanoliposomes. METHODS: Nanoliposomes and metallochelating nanoliposomes were prepared by lipid film hydration and extrusion methods. The structure of the liposomal formulation was studied by electron microscopy, AF microscopy, and dynamic light scattering. Sublethal and lethal γ-irradiation mice models were used to demonstrate stimulation of innate immune system. Recombinant Hsp90 antigen (Candida albicans) bound onto metallochelating nanoliposomes was used for immunisation of mice to demonstrate adjuvant activities of tested compounds. RESULTS: Safety and stimulation of innate and adaptive immunity were demonstrated on rabbits and mice. The liposomal formulation of norAbuMDP/GMDP was apyrogenic in rabbit test and lacking any side effect in vivo. Recovery of bone marrow after sublethal γ-irradiation as well as increased survival of mice after lethal irradiation was demonstrated. Enhancement of specific immune response was demonstrated for some derivatives incorporated in metallochelating nanoliposomes with recombinant Hsp90 protein antigen. CONCLUSIONS: Liposomal formulations of new lipophilic derivatives of norAbuMDP/GMDP proved themselves as promising adjuvants for recombinant vaccines as well as immunomodulators for stimulation of innate immunity and bone-marrow recovery after chemo/radio therapy of cancer.

The Superior Effect of Radiofrequency With Targeted Ultrasound for Facial Rejuvenation by Inducing Hyaluronic Acid Synthesis: A Pilot Preclinical Study.

Background: The level of dermal hyaluronic acid (HA) can be depleted by 75% at age 70. HA provides dermal hydration, volume, and thickness, making it a major component of the extracellular matrix. Restoration of dermal and epidermal HA can be achieved by combining radiofrequency (RF) energy and targeted ultrasound (TUS). The monopolar RF generates heat, with the TUS stimulating HA production. The heat induces a regenerative response in the skin, increasing the fibroblast activity and producing various extracellular matrix compounds, including HA. Objectives: To investigate the effect of the simultaneous application of RF + TUS or RF + US on the stimulation of HA production. Methods: Twelve animals underwent 4 treatments. Six were treated with transcutaneous RF + TUS and 6 with the combination RF + US. The opposite untreated side served as a control. Punch biopsies of the skin were taken at baseline, immediately posttreatment, 1 month, and 2 months posttreatment. The tissue was evaluated with real-time quantitative polymerase chain reaction (RT-qPCR), matrix-assisted laser desorption (MALDI) and time of flight (TOF), and confocal microscopy. Results: The RT-qPCR focused on assessing the production of has1 and has2, enzymes responsible for HA synthesis. RT-qPCR results of the RF + TUS group revealed a +98% and +45% increase in hyaluronic synthetase (HAS) 1 and HAS2 production after the treatments, respectively. The MALDI-TOF revealed a +224% increase in measured HA 2 months after the treatments. The changes were also visible in the confocal microscopy. The control group showed no significant (P > .05) results in either of the evaluation methods. Conclusions: Concurrent application of RF and TUS significantly enhances the natural regenerative processes in skin tissue.

Enhancing Solubility and Bioefficacy of Stilbenes by Liposomal Encapsulation-The Case of Macasiamenene F.

Stilbenes in food and medicinal plants have been described as potent antiphlogistic and antioxidant compounds, and therefore, they present an interesting potential for the development of dietary supplements. Among them, macasiamenene F (MF) has recently been shown to be an effective anti-inflammatory and cytoprotective agent that dampens peripheral and CNS inflammation in vitro. Nevertheless, this promising molecule, like other stilbenes and a large percentage of drugs under development, faces poor water solubility, which results in trickier in vivo administration and low bioavailability. With the aim of improving MF solubility and developing a form optimized for in vivo administration, eight types of conventional liposomal nanocarriers and one type of PEGylated liposomes were formulated and characterized. In order to select the appropriate form of MF encapsulation, the safety of MF liposomal formulations was evaluated on THP-1 and THP-1-XBlue-MD2-CD14 monocytes, BV-2 microglia, and primary cortical neurons in culture. Furthermore, the cellular uptake of liposomes and the effect of encapsulation on MF anti-inflammatory effectiveness were evaluated on THP-1-XBlue-MD2-CD14 monocytes and BV-2 microglia. MF (5 mol %) encapsulated in PEGylated liposomes with an average size of 160 nm and polydispersity index of 0.122 was stable, safe, and the most promising form of MF encapsulation keeping its cytoprotective and anti-inflammatory properties.

Mitochondrial targeting overcomes ABCA1-dependent resistance of lung carcinoma to α-tocopheryl succinate.

α-Tocopheryl succinate (α-TOS) is a promising anti-cancer agent due to its selectivity for cancer cells. It is important to understand whether long-term exposure of tumour cells to the agent will render them resistant to the treatment. Exposure of the non-small cell lung carcinoma H1299 cells to escalating doses of α-TOS made them resistant to the agent due to the upregulation of the ABCA1 protein, which caused its efflux. Full susceptibility of the cells to α-TOS was restored by knocking down the ABCA1 protein. Similar resistance including ABCA1 gene upregulation was observed in the A549 lung cancer cells exposed to α-TOS. The resistance of the cells to α-TOS was overcome by its mitochondrially targeted analogue, MitoVES, that is taken up on the basis of the membrane potential, bypassing the enhanced expression of the ABCA1 protein. The in vitro results were replicated in mouse models of tumours derived from parental and resistant H1299 cells. We conclude that long-term exposure of cancer cells to α-TOS causes their resistance to the drug, which can be overcome by its mitochondrially targeted counterpart. This finding should be taken into consideration when planning clinical trials with vitamin E analogues.

Development of 3D Printed Multi-Layered Orodispersible Films with Porous Structure Applicable as a Substrate for Inkjet Printing.

The direct tailoring of the size, composition, or number of layers belongs to the advantages of 3D printing employment in producing orodispersible films (ODFs) compared to the frequently utilized solvent casting method. This study aimed to produce porous ODFs as a substrate for medicated ink deposited by a 2D printer. The innovative semi-solid extrusion 3D printing method was employed to produce multilayered ODFs, where the bottom layer assures the mechanical properties. In contrast, the top layer provides a porous structure for ink entrapment. Hydroxypropyl methylcellulose and polyvinyl alcohol were utilized as film-forming polymers, glycerol as a plasticizer, and sodium starch glycolate as a disintegrant in the bottom matrix. Several porogen agents (Aeroperl® 300, Fujisil®, Syloid® 244 FP, Syloid® XDP 3050, Neusilin® S2, Neusilin® US2, and Neusilin® UFL2) acted as porosity enhancers in the two types of top layer. ODFs with satisfactory disintegration time were prepared. The correlation between the porogen content and the mechanical properties was proved. A porous ODF structure was detected in most samples and linked to the porogen content. SSE 3D printing represents a promising preparation method for the production of porous ODFs as substrates for subsequent drug deposition by 2D printing, avoiding the difficulties arising in casting or printing medicated ODFs directly.

UV-C irradiation as an effective tool for sterilization of porcine chimeric VP1-PCV2bCap recombinant vaccine.

Ultraviolet irradiation is an effective method of virus and bacteria inactivation. The dose of UV-C light necessary for baculovirus inactivation by measurement of fluorescent GFP protein produced by baculovirus expression system after the irradiation of baculovirus culture in doses ranging from 3.5 to 42 J/m2 was determined. At a dose of 36.8 J/m2, only 0.5% of GFP-expressing cells were detected by flow cytometry and confocal microscopy. The stability of purified VP1-PCV2bCap protein produced by baculovirus expression system was analyzed after the irradiation at doses ranging from 3.5 to 19.3 J/m2. Up to the dose of 11 J/m2, no significant effect of UV-C light on the stability of VP1-PCV2bCap was detected. We observed a dose-dependent increase in VP1-PCV2bCap-specific immune response in BALB/c mice immunized by recombinant protein sterilized by irradiation in dose 11 J/m2 with no significant difference between vaccines sterilized by UV-C light and filtration. A substantial difference in the production of VP1-PCV2bCap specific IgG was observed in piglets immunized with VP1-PCV2bCap sterilized by UV-C in comparison with protein sterilized by filtration in combination with the inactivation of baculovirus by binary ethylenimine. UV-C irradiation represents an effective method for vaccine sterilization, where commonly used methods of sterilization are not possible.

Development of modern immunization agent against bovine papillomavirus type 1 infection based on BPV1 L1 recombinant protein.

Bovine papillomavirus type 1 L1 protein was produced in a baculovirus expression system and purified as virus-like particles (VLPs) by affinity chromatography using lectins. The morphological integrity of VLPs was confirmed by electron microscopy. Differences between the two detected variants were deciphered by mass spectrometry of peptides (MALDI-TOF). Mice were immunized with purified VLPs in doses of 10, 25, or 50 µg in combination with 1% saponin and 15% alhydrogel per dose as adjuvants. Analysis of the humoral immune response revealed increased levels of specific antibodies detected 3 weeks after the first immunization in all groups of animals. This was further significantly increased by the booster applied 3 weeks after the first dose, with the best immune response in a group of mice immunized by the largest dose of antigen. BPV1 L1 VLPs purified by affinity chromatography using lectins could be used for prophylactic immunization in veterinary medicine.

Antiviral activity of singlet oxygen-photogenerating perylene compounds against SARS-CoV-2: Interaction with the viral envelope and photodynamic virion inactivation.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has prompted great interest in novel broad-spectrum antivirals, including perylene-related compounds. In the present study, we performed a structure-activity relationship analysis of a series of perylene derivatives, which comprised a large planar perylene residue, and structurally divergent polar groups connected to the perylene core by a rigid ethynyl or thiophene linker. Most of the tested compounds did not exhibit significant cytotoxicity towards multiple cell types susceptible to SARS-CoV-2 infection, and did not change the expressions of cellular stress-related genes under normal light conditions. These compounds showed nanomolar or sub-micromolar dose-dependent anti-SARS-CoV-2 activity, and also suppressed the in vitro replication of feline coronavirus (FCoV), also termed feline infectious peritonitis virus (FIPV). Perylene compounds exhibited high affinity for liposomal and cellular membranes, and efficiently intercalated into the envelopes of SARS-CoV-2 virions, thereby blocking the viral-cell fusion machinery. Furthermore, the studied compounds were demonstrated to be potent photosensitizers, generating reactive oxygen species (ROS), and their anti-SARS-CoV-2 activities were considerably enhanced after irradiation with blue light. Our results indicated that photosensitization is the major mechanism underlying the anti-SARS-CoV-2 activity of perylene derivatives, with these compounds completely losing their antiviral potency under red light. Overall, perylene-based compounds are broad-spectrum antivirals against multiple enveloped viruses, with antiviral action based on light-induced photochemical damage (ROS-mediated, likely singlet oxygen-mediated), causing impairment of viral membrane rheology.

Exploration of Neusilin® US2 as an Acceptable Filler in HPMC Matrix Systems-Comparison of Pharmacopoeial and Dynamic Biorelevant Dissolution Study.

Modern pharmaceutical technology still seeks new excipients and investigates the further use in already known ones. An example is magnesium aluminometasilicate Neusilin® US2 (NEU), a commonly used inert filler with unique properties that are usable in various pharmaceutical fields of interest. We aimed to explore its application in hypromellose matrix systems (HPMC content 10-30%) compared to the traditionally used microcrystalline cellulose (MCC) PH 102. The properties of powder mixtures and directly compressed tablets containing individual fillers NEU or MCC, or their blend with ratios of 1.5:1, 1:1, and 0.5:1 were investigated. Besides the routine pharmaceutical testing, we have enriched the matrices' evaluation with a biorelevant dynamic dissolution study and advanced statistical analysis. Under the USP apparatus 2 dissolution test, NEU, individually, did not provide advantages compared to MCC. The primary limitations were the burst effect increase followed by faster drug release at the 10-20% HPMC concentrations. However, the biorelevant dynamic dissolution study did not confirm these findings and showed similarities in dissolution profiles. It indicates the limitations of pharmacopoeial methods in matrix tablet development. Surprisingly, the NEU/MCC blend matrices at the same HPMC concentration showed technologically advantageous properties. Besides improved flowability, tablet hardness, and a positive impact on the in vitro drug dissolution profile toward zero-order kinetics, the USP 2 dissolution data of the samples N75M50 and N50M50 showed a similarity to those obtained from the dynamic biorelevant apparatus with multi-compartment structure. This finding demonstrates the more predictable in vivo behaviour of the developed matrix systems in human organisms.

Rational Design of Self-Emulsifying Pellet Formulation of Thymol: Technology Development Guided by Molecular-Level Structure Characterization and Ex Vivo Testing.

The growing need for processing natural lipophilic and often volatile substances such as thymol, a promising candidate for topical treatment of intestinal mucosa, led us to the utilization of solid-state nuclear magnetic resonance (ss-NMR) spectroscopy for the rational design of enteric pellets with a thymol self-emulsifying system (SES). The SES (triacylglycerol, Labrasol®, and propylene glycol) provided a stable o/w emulsion with particle size between 1 and 7 µm. The ex vivo experiment confirmed the SES mucosal permeation and thymol delivery to enterocytes. Pellets W90 (MCC, Neusilin®US2, chitosan) were prepared using distilled water (90 g) by the M1−M3 extrusion/spheronisation methods varying in steps number and/or cumulative time. The pellets (705−740 µm) showed mostly comparable properties­zero friability, low intraparticular porosity (0−0.71%), and relatively high density (1.43−1.45%). They exhibited similar thymol release for 6 h (burst effect in 15th min ca. 60%), but its content increased (30−39.6 mg/g) with a shorter process time. The M3-W90 fluid-bed coated pellets (Eudragit®L) prevented undesirable thymol release in stomach conditions (<10% for 3 h). A detailed, ss-NMR investigation revealed structural differences across samples prepared by M1−M3 methods concerning system stability and internal interactions. The suggested formulation and methodology are promising for other lipophilic volatiles in treating intestinal diseases.

Cytokine expression by CD163+ monocytes in healthy and Actinobacillus pleuropneumoniae-infected pigs.

Distinct monocyte subpopulations have been previously described in healthy pigs and pigs experimentally infected with Actinobacillus pleuropneumoniae (APP). The CD163+ subpopulation of bone marrow (BM), peripheral blood (PB) and lung monocytes was found to play an important role in the inflammatory process. The inflammation is accompanied by elevation of inflammatory cytokines. The aim of the study was to evaluate the contribution of CD163+ monocytes and macrophages to cytokine production during APP-induced lung inflammation. Cytokine production was assessed by flow cytometry (FC) and quantitative PCR (qPCR) in CD163+ monocytes and by qPCR, immunohistochemistry/fluorescence in lungs and tracheobronchial lymph nodes (TBLN). Despite the systemic inflammatory response after APP infection, BM and PB CD163+ monocytes did not express elevated levels of a wide range of cytokines compared to control pigs. In contrast, significant amounts of IL-1ß, IL-6, IL-8 and TNF-α were produced in lung lesions and IL-1ß in the TBLN. At the protein level, TNF-α was expressed by both CD163+ monocytes and macrophages in lung lesions, whereas IL-1ß, IL-6 and IL-8 expression was found only in CD163+ monocytes; no CD163+ macrophages were found to produce these cytokines. Furthermore, the quantification of CD163+ monocytes expressing the two cytokines IL-1ß and IL-8 that were most elevated was performed. In lung lesions, 36.5% IL-1ß positive CD163+ monocytes but only 18.3% IL-8 positive CD163+ monocytes were found. In conclusion, PB and BM CD163+ monocytes do not appear to contribute to the elevated cytokine levels in plasma. On the other hand, CD163+ monocytes contribute to inflammatory cytokine expression, especially IL-1ß at the site of inflammation during the inflammatory process.

Immobilization of histidine-tagged proteins on monodisperse metallochelation liposomes: Preparation and study of their structure.

Liposomes represent a biocompatible platform for the construction of self-assembling proteoliposomes using nickel or zinc metallochelation. Potential applications of such structures consist in the development of new biocompatible vaccination nanoparticles and drug delivery nanoparticle systems. Here, we describe the design and construction of a flow-through ultrafiltration cell suitable for the preparation of monodisperse liposomes enabled for metallochelation and, hence, the formation of proteoliposomes. The linkage of the cell with a fast protein liquid chromatography system facilitates automation of the procedure, which fits the criteria for upscaling. Proof-of-concept experiments are performed using a mixture of egg phosphatidyl choline and nickel-chelating lipid DOGS-NTA-Ni (1,2-dioleoyl-sn-glycero-3-{[N(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl}(nickel salt)) to formulate proteoliposomes with proteins attached by metallochelation, including histidine (His)-tagged recombinant green fluorescent protein and rgp120 (derived from HIV-1 Env). These model proteoliposomes are characterized by gel permeation chromatography and by dynamic light scattering. Transmission electron microscopy and immunogold staining are used to characterize surface-bound proteins, revealing the tendency of rgp120 to form microdomains on liposome surfaces. These microdomains possess a two-dimensional crystal-like structure that is seen more precisely by atomic force microscopy.