Salt-Induced Adsorption and Rupture of Liposomes on Microplastics.

Microplastics have attracted considerable attention because of concerns regarding their environmental risks to living systems. The interaction between the lipid bilayer and microplastics is important for examining the potential harm to biological membranes in the presence of microplastics. In addition, membrane coatings may change the surface and colloidal properties of microplastics. Herein, phosphatidylcholine (PC) lipids, whose headgroup is most common in cell membranes, were used as model lipids. The adsorption and rupture of PC liposomes on microplastics were systematically studied. We found that divalent metal ions, such as Mg2+ and Ca2+, facilitate liposome adsorption onto microplastics and induce 40-55% liposome leakage at 2.5 mM. In contrast, to achieve a similar effect, 300 mM Na+ was required. Adsorption and rupture followed the same metal concentration requirements, suggesting that liposome adsorption was the rate-limiting step. After adsorption with liposomes, microplastics became more hydrophilic and were better dispersed in water. A similar behavior was observed for all five types of tested microplastics, including PP, PE, PVC, PET, and PS. Leakage also occurred in ocean water. This study provides fundamental insights into the interactions between liposomes and microplastics and has implications for the colloidal and transport properties of microplastics.

Spherical DNA for Probing Wettability of Microplastics.

Wettability of microplastics may change due to chemical or physical transformations at their surface. In this work, we studied the adsorption of spherical nucleic acids (SNAs) with a gold nanoparticle core and linear DNA of the same sequence to probe the wettability of microplastics. Soaking microplastics in water at room temperature for 3 months resulted in the enhancement of SNA adsorption capacity and affinity, whereas linear DNA adsorption was the same on the fresh and soaked microplastics. Drying of the soaked microplastics followed by rehydration decreased the adsorption of the SNA, suggesting that the effect of soaking was reversible and related to physical changes instead of chemical changes of the microplastics. Raman spectroscopy data also revealed no chemical transformations of the soaked microplastics. Heating of microplastics over a short period induced a similar effect to long-term soaking. We propose that soaking or heating removes air entrapped in the nanosized pores at the water-plastic interface, increasing the contact surface area of the SNA to afford stronger adsorption. However, such wetted porosity would not change the adsorption of linear DNA because of its much smaller size.

General Label-Free Fluorescent Aptamer Binding Assay Using Cationic Conjugated Polymers.

With more and more new aptamers being reported, a general, cost-effective yet reliable aptamer binding assay is still needed. Herein, we studied cationic conjugated polymer (CCP)-based binding assays taking advantage of the conformational change of aptamer after binding with a target, which is reflected by the fluorescence change of the CCP. Poly(3-(3'-N,N,N-triethylamino-1'-propyloxy)-4-methyl-2,5-thiophene hydrochloride) (PMNT) was used as a model CCP in this study, and the optimal buffer was close to physiological conditions with 100 mM NaCl and 10 mM MgCl2. We characterized four aptamers for K+, adenosine, cortisol, and caffeine. For cortisol and caffeine, the drop in the 580 nm peak intensity was used for quantification, whereas for K+ and adenosine, the fluorescence ratio at 580 over 530 nm was used. The longer stem of the stem-loop structured aptamer facilitated binding of the target and enlarged the detection signal. High specificity was achieved in differentiating targets with analogues. Compared with the SYBR Green I dye-based staining method, our method achieved equal or even higher sensitivity. Therefore, this assay is practicable as a general aptamer binding assay. The simple, label-free, quick response, and cost-effective features will make it a useful method to evaluate aptamer binding. At the same time, this system can also serve as label-free biosensors for target detection.

Adsorption of Linear and Spherical DNA Oligonucleotides onto Microplastics.

Microplastic pollution of water and food chains can endanger human health. It has been reported that environmental DNA can be carried by microplastics and spread into the ecosystem. To better comprehend the interactions between microplastics and DNA, we herein investigated the adsorption of DNA oligonucleotides on a few important microplastics. The microplastics were prepared using common plastic objects made of polyethylene (PE), polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC), composite of PS/PVC, and polyethylene terephthalate (PET). The effect of environmentally abundant metal ions such as Na+, Mg2+, and Ca2+ on the adsorption was also studied. Among the microplastics, PET and PS had the highest efficiency for the adsorption of linear DNA, likely due to the interactions provided by their aromatic rings. The study of DNA desorption from PET revealed the important role of hydrogen bonding and metal-mediated adsorption, while van der Waals force and hydrophobic interactions were also involved in the adsorption mechanism. The adsorption of spherical DNA (SNA) made of a high density of DNA coated on gold nanoparticles (AuNPs) was also studied, where the adsorption affinity order was found to be PET > PS/PVC > PS. Moreover, a tighter DNA adsorption was achieved in the presence of Ca2+ and Mg2+ compared to Na+.

Removal and Degradation of Microplastics Using the Magnetic and Nanozyme Activities of Bare Iron Oxide Nanoaggregates.

Removal and degradation of microplastics are often carried out separately. In this work, hydrophilic bare Fe3 O4 nanoaggregates allowed efficient removal of the most common microplastics including high-density polyethylene, polypropylene, polyvinyl chloride, polystyrene, and polyethylene terephthalate. Full extraction was achieved using Fe3 O4 at 1 % of the mass of microplastics. Hydrogen bonding is the main force for the adsorption of Fe3 O4 . Unlike the more commonly used hydrophobically modified Fe3 O4 nanoparticles, the bare Fe3 O4 benefitted from the peroxidase-like activity of its exposed surface, enabling further catalytic degradation of microplastics with nearly 100 % efficiency and easy recovery of the Fe3 O4 .

A Polymeric Nanobeacon for Monitoring the Fluctuation of Hydrogen Polysulfides during Fertilization and Embryonic Development.

Fertilization and early embryonic development as the beginning of a new life are key biological events. Hydrogen polysulfide (H2 Sn ) plays important roles during physiological regulation, such as antioxidation-protection. However, no report has studied in situ H2 Sn fluctuation during early embryonic development because of the low abundance of H2 Sn and inadequate sensitivity of probes. We herein construct a polymeric nanobeacon from a H2 Sn -responsive polymer and fluorophores, which is capable of detecting H2 Sn selectively and of signal amplification. Taking the zebrafish as a model, the polymeric nanobeacon revealed that the H2 Sn level was significantly elevated after fertilization due to the activation of cell multiplication, suppressed partially during embryonic development, and finally kept steady up to zebrafish emergence. This strategy is generally accessible for biomarkers by altering the responsive unit and significant for facilitating biological analysis during life development.

Enhancing the Sensitivity of DNA and Aptamer Probes in the Dextran/PEG Aqueous Two-Phase System.

Increasing the local concentration of DNA-based probes is a convenient way to improve the sensitivity of biosensors. Instead of using organic solvents or ionic liquids that phase-separate with water based on hydrophobic interactions, we herein studied a classic aqueous two-phase system (ATPS) comprising polyethylene glycol (PEG) and dextran. Polymers of higher molecular weights and higher concentrations favored phase separation. DNA oligonucleotides are selectively enriched in the dextran-rich phase unless the pH was increased to 12. A higher volume ratio of PEG-to-dextran and a higher concentration of PEG also enrich more DNA probes in the dextran-rich phase. The partition efficiency of the T15 DNA was enriched around seven times in the dextran phase when the volume ratio of dextran and PEG reached 1:10. The detection of limit improved by 3.6-fold in a molecular beacon-based DNA detection system with the ATPS. The ATPS also increased the sensitivity for the detection of Hg2+ and adenosine triphosphate, although these target molecules alone distributed equally in the two phases. This work demonstrates a simple method using water soluble polymers to improve biosensors.

Spherical Nucleic Acid Mediated Functionalization of Polydopamine-Coated Nanoparticles for Selective DNA Extraction and Detection.

Magnetic nanoparticles have been widely used for the separation of biomolecules for biological applications due to the mild and efficient separation process. In previous studies, core-shell magnetic nanoparticles (NPs) were designed for DNA extraction without much sequence specificity. In this work, to achieve highly selective DNA extraction, we designed a core-shell magnetic structure by coating polydopamine (PDA) on Fe3O4 NPs. Without divalent metal ions, PDA does not adsorb DNA at neutral pH. The Fe3O4@PDA NPs were then functionalized with spherical nucleic acids (SNA) to provide a high density of probe DNA. Fe3O4@PDA@SNA was also compared with when a linear SH-DNA was covalently attached to the NPs surface, showing a higher density of the probe SNA than SH-DNA can be loaded on the NPs in a remarkably shorter time. Nonspecific DNA extraction was thoroughly inhibited by both probes. DNA extraction by the Fe3O4@PDA@SNA was more effective as well as 5-fold faster than by the Fe3O4@PDA@SH-DNA, probably due to the favorable standing conformation of DNA strands in SNA. Moreover, extraction by Fe3O4@PDA@SNA showed high robustness in fetal bovine serum, and the same design can be used for selective detection of DNA. Finally, the method was also demonstrated on silica NPs and WS2 nanosheets for coating with PDA and SNA. Altogether, our findings revealed an interesting and general surface modification strategy using PDA@SNA conjugates for sequence-specific DNA extraction.

Metal-Doped Polydopamine Nanoparticles for Highly Robust and Efficient DNA Adsorption and Sensing.

Controlling DNA adsorption on nanomaterials is crucial for a wide range of applications in analytical and biomedical sciences. Polydopamine (PDA) is a versatile material that can be coated on nearly any surface, and thus adsorbing DNA onto PDA can be a general method for indirect DNA functionalization of surfaces. Polyvalent metal ions were reported to promote DNA adsorption on PDA nanoparticles (NPs), but previous works added the metal ions after the formation of PDA. Herein, we compared the effect of polyvalent metal ions added during the synthesis of PDA NPs (called metal-doped) with the effect of polyvalent metal ions added after the synthesis (metal-adsorbed). A series of metal ions including Ca2+, Zn2+, Ni2+, Fe3+, and Gd3+ were tested, and Zn2+ was studied in detail due to its excellent ability for promoting DNA adsorption. With 100 µM Zn2+, metal-doped NPs were ∼30% more efficient than metal-adsorbed NPs for DNA adsorption in buffer attributable to a higher metal loading on the surface of the metal-doped NPs. Metal leaching was negligible from the metal-doped NPs, and they showed a remarkably higher robustness than the metal-adsorbed NPs, resulting in a 20-fold higher DNA extraction efficiency from serum. Based on the desorption studies, a higher adsorption affinity for the metal-doped NPs was confirmed. Finally, the Zn2+-doped PDA NPs were used for sensitive DNA detection with a limit of detection of 0.45 nM, and the sensor was highly resistant to nonspecific protein and phosphate displacement.

Liposome-Boosted Peroxidase-Mimicking Nanozymes Breaking the pH Limit.

Peroxidase-mimicking nanozymes such as Fe3 O4 nanoparticles are promising substitutes for natural enzymes like horseradish peroxidase. However, most such nanozymes work efficiently only in acidic conditions. In this work, the influence of various liposomes on nanozyme activity was studied. By introducing negatively charged liposomes, peroxidase-mimicking nanozymes achieved oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in neutral and even alkaline conditions, although the activity towards anionic 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was inhibited. The Fe3 O4 nanoparticles adsorbed on the liposomes without disrupting membrane integrity as confirmed by fluorescence quenching, dye leakage assays, and cryo-electron microscopy. Stabilization of the blue-colored oxidized products of TMB by electrostatic interactions was believed to be the reason for the enhanced activity. This work has introduced lipids to nanozyme research, and it also has practically important applications for using nanozymes at neutral pH, such as the detection of hydrogen peroxide and glucose.

Transition Metal-Mediated DNA Adsorption on Polydopamine Nanoparticles.

Polydopamine (PDA) is a widely used universal coating for a broad range of materials. Interfacing PDA with various biomolecules, such as DNA, is critical for applications such as sensing, intracellular delivery, and material fabrication. Because of the negative surface charge of PDA at neutral pH, electrostatic repulsion exists between PDA and DNA. In previous studies, modified DNA or low pH was used to overcome this repulsion for DNA adsorption. More recently, divalent Ca2+ was found to bridge DNA and PDA. Herein, we studied four transition metals (Mn2+, Co2+, Zn2+, and Ni2+) and compared their efficiencies with Ca2+ for promoting DNA adsorption. These transition metals induced a more efficient and tighter DNA binding compared to Ca2+. In all these cases, the DNA phosphate backbone played a dominant role in adsorption, although DNA bases might also interact with strong binding metals such as Ni2+. Moreover, when the adsorption affinity was stronger, sensing was more selective to complementary DNA. Finally, aging of PDA appeared to be detrimental for DNA adsorption, which could be due to further oxidation of PDA. We showed that using Zn2+ or Ni2+ could considerably relieve the aging effect, while storing PDA at 4 °C could slow down aging.

Molecular Imprinting with Functional DNA.

Molecular imprinting refers to templated polymerization with rationally designed monomers, and this is a general method to prepare stable and cost-effective ligands. This attractive concept however suffers from low affinity, low specificity, and limited signaling mechanisms for binding. Acrydite-modified DNA oligonucleotides can be readily copolymerized into acrylic polymers. With molecular recognition and catalytic functions, such functional DNAs are recently shown to enhance the performance of molecularly imprinted polymers (MIPs) in a few ways. First, DNA aptamers are used as macromonomers to enhance binding affinity and specificity of MIPs. Second, DNA can help produce optical signals to follow binding events. Third, imprinting can also improve the performance of catalytic DNA by enhancing its activity and specificity toward the template substrate. Finally, MIP is shown to help aptamer selection. Bulk imprinting, nanoparticle imprinting, and surface imprinting are all demonstrated with DNA. Since both DNA and synthetic polymers are cost effective and stable, their hybrid materials still possess such properties while enhancing the function of each component. This review covers recent developments on the abovementioned aspects of DNA-containing MIPs, a field just emerged in the last five years, and future research directions are discussed toward the end.

Growing a Nucleotide/Lanthanide Coordination Polymer Shell on Liposomes.

Coating liposomes with a shell is a useful strategy to increase membrane stability and prevent leakage or fusion. Nucleotide/lanthanide coordination nanoparticles (NPs) are formed by a simple mixing at ambient conditions. Because some lipid headgroups contain lanthanide binding ligands, they may direct the growth of such coordination NPs. Herein, a gadolinium/adenosine monophosphate (Gd3+/AMP) shell was formed on liposomes (liposome@Gd3+/AMP) using lipids containing phosphoserine (PS) or cholinephosphate (CP) headgroups, while phosphocholine liposomes did not support the shell. Liposome binding Gd3+ is confirmed by transmission electron microscopy (TEM). The negatively charged CP and PS liposomes reversed to positive upon Gd3+ binding, while other metals such as Ca2+ and Zn2+ did not reverse the charge. Binding of Gd3+ did not leak the PS liposomes. Then, AMP was further added to cross-link Gd3+ on the liposome surface. A shell was formed as indicated by TEM, and the content inside the liposome remained for the PS liposomes. While adding Triton X-100 still induced leakage of the encapsulated liposomes, the shell protected the liposomes from leakage induced by ZnO NPs, suggesting a porous structure of the Gd3+/AMP shell which allowed penetration of Triton X-100 but not the larger ZnO NPs. This work provides a simple method to coat liposomes, and also offers a fundamental understanding of liposome adsorption of lanthanide ions.

Fluorescence Polarization for Probing DNA Adsorption by Nanomaterials and Fluorophore/DNA Interactions.

Fluorescence polarization (FP) is attractive for measuring binding interactions and has been recently used to study DNA adsorption on nanomaterials. Since most nanomaterials are strong fluorescence quenchers, correlations among adsorption efficiency, quenching efficiency, and FP need to be interpreted carefully. In this work, carboxyfluorescein (FAM)-labeled DNA oligonucleotides were studied under various quenching conditions. First, quenching was induced by lowering the pH, taking advantage of the fact that FAM is almost nonfluorescent at a pH below 4. Strong interactions were observed between the FAM label and polyadenine DNA, as judged by the increased FP at low pH, while FAM-labeled polythymine DNA was less affected by the pH. Comparisons were also performed with FAM-labeled poly(ethylene glycol) and bovine serum albumin. An equation was derived to calculate the effect of fluorescence quenching and DNA adsorption by nanomaterials. For strongly quenching nanomaterials, such as graphene oxide, DNA adsorption alone does not change the measured FP. Light scattering and weak fluorescence from graphene oxide increase FP in these cases. For comparison, a strongly adsorbing but weak quenching material, Y2O3, was also studied and the result was consistent with a normal binding reaction. Overall, FP is a powerful technique for binding and adsorption assays, but quenched samples need to be interpreted with care.

Charge and Coordination Directed Liposome Fusion onto SiO2 and TiO2 Nanoparticles.

TiO2 and SiO2 are very useful materials for building biointerfaces. A particularly interesting aspect is their interaction with lipid bilayers. Many past research efforts focused on phosphocholine (PC) lipids, which form supported lipid bilayers (SLB) on SiO2 at physiological conditions but are adsorbed as intact liposomes on TiO2. Low pH was required to form PC SLBs on TiO2. This work intends to understand the surface forces and chemistry responsible for such differences. Two charge neutral lipids: 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC) and 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl ethyl phosphate (DOCPe) and two negatively charged lipids: 1,2-dioleoyl- sn-glycero-3-phospho-l-serine (DOPS) and 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl hydrogen phosphate (DOCP) were used. Using calcein leakage assays, adsorption measurement, cryo-TEM, and washing, we concluded that charge is the dominating factor on SiO2. The two neutral lipids form SLB on SiO2 at pH 3 and 7, but the two negatively charged ones cannot form. On TiO2, both charge and coordination chemistry are important. The two anionic lipids formed SLB from pH 3 to 10. DOCP had stronger affinity than DOPS likely due to the tighter terminal phosphate binding of the former. The two neutral liposomes formed SLB only at pH 3, where phosphate interaction and van der Waals force are deemed important. The pH 3 prepared TiO2 DOPC SLBs are destabilized at neutral pH, indicating the reversible nature of the interaction. This work has provided new insights into two important materials interacting with common liposomes, which are important for reproducible biosensing, device fabrication, and drug delivery applications.

DNA Oligonucleotide-Functionalized Liposomes: Bioconjugate Chemistry, Biointerfaces, and Applications.

Interfacing DNA with liposomes has produced a diverse range of programmable soft materials, devices, and drug delivery vehicles. By simply controlling liposomal composition, bilayer fluidity, lipid domain formation, and surface charge can be systematically varied. Recent development in DNA research has produced not only sophisticated nanostructures but also new functions including ligand binding and catalysis. For noncationic liposomes, a DNA is typically covalently linked to a hydrophobic or lipid moiety that can be inserted into lipid membranes. In this article, we discuss fundamental biointerfaces formed between DNA and noncationic liposomes. The methods to prepare such conjugates and the interactions at the membrane interfaces are also discussed. The effect of DNA lateral diffusion on fluid bilayer membranes and the effect of membrane on DNA assembly are emphasized. DNA hybridization can be programmed to promote fusion of lipid membranes. Representative applications of this conjugate for drug delivery, biosensor development, and directed assembly of materials are briefly described toward the end. Some future research directions are also proposed to further understand this biointerface.

Cu2+-Directed Liposome Membrane Fusion, Positive-Stain Electron Microscopy, and Oxidation.

Natural lipid headgroups contain a few types of metal ligands, such as phosphate, amine, and serine, which interact with metal ions differently. Herein, we studied the binding between Cu2+ and liposomes with four types of headgroups: phosphocholine (PC), phosphoglycerol (PG), phosphoserine (PS), and cholinephosphate (CP). Using fluorescently headgroup-labeled liposomes, Cu2+ strongly quenched the CP and PS liposomes, whereas quenching of PC and PG was weaker. Dynamic light scattering indicated that all of the four liposomes aggregated at high Cu2+ concentrations, and ethylenediaminetetraacetic acid (EDTA) only restored the original size of the PC liposome, implying fusion of the other three types of liposomes. The leakage tests revealed that the integrity of PC liposomes was not affected by Cu2+, but the other three liposomes leaked. Under TEM, all of the liposomes show a positive-stain feature in the presence of Cu2+ and Cu2+-stained individual liposomes with a short incubation time (<1 min). The oxidative catalytic property of Cu2+ was also tested, and a tight binding by the PS liposome inhibited the activity of Cu2+. Finally, a model of interaction for each liposome was proposed, and each one has a different metal-binding and interaction mechanism.

Headgroup-Inversed Liposomes: Biointerfaces, Supported Bilayers and Applications.

Phospholipids are a major component of the cell membrane. In most natural phospholipids, the phosphate acts as a bridge, connecting the other portion of the polar headgroup with the hydrophobic tails. Such bridging phosphate is chemically quite inert. Synthetic lipids inversing the headgroup polarity of phosphocholine (PC) have been recently reported, and these are named CP lipids with a terminal phosphate, or CPe with the terminal phosphate capped by an ethyl group. This Feature Article summarizes the properties and applications of such inversed lipids. First, CPe liposomes were found to be highly resistant to protein adsorption with an even longer blood circulation time than PC liposomes, allowing for enhanced accumulation in tumor sites. CPe liposomes do not interact with PC liposomes either, and this observation was different from that reported using CP polymers, which adhere strongly to cells. Second, CP liposomes interact strongly with many metal oxide nanoparticles (but not silica) forming supported lipid bilayers, while PC liposomes only form supported bilayers on silica. Finally, CP liposomes are good metal ligands based on their exposed terminal phosphate. Zn2+ binds to CP liposomes so strongly that Zn2+ sandwiched multilayered lipid structures were observed. Aside from these fundamental aspects, the potential applications of these headgroup-inversed lipids in drug delivery and biosensor development have also been described, which in turn has promoted fundamental biointerface insights.

Sub-Angstrom Gold Nanoparticle/Liposome Interfaces Controlled by Halides.

A hallmark of nanoscience is size-dependent and distance-dependent physical properties. Although most previous studies focused on optical properties, which are often tuned at nanometer scale, we herein report on the interaction between halide-capped gold nanoparticles (AuNPs) and phosphocholine (PC) liposomes at the sub-Angstrom level. Halide-capped AuNPs are adsorbed by PC liposomes attributable to van der Waals force. Iodide-capped AuNPs interact much more weakly with the liposomes compared with bromide- and chloride-capped AuNPs, as indicated by a liposome leakage assay and differential scanning calorimetry. This is explained by the slightly larger size of iodide separating the AuNP core more from the liposome surface. Cryo-transmission electron microscopy indicates that the liposomes remain intact when mixed with these halide-capped AuNPs of 13 or 70 nm in diameter. Other even larger ligands, including small thiol compounds, DNA oligonucleotides, proteins, and polymers, fully blocked the interaction, whereas AuNPs dispersed in noninteracting ions, including fluoride, phosphate, perchlorate, nitrate, sulfate, and bicarbonate, are still adsorbed strongly by 1,2-dioleoyl- sn-glycero-3-phosphocholine liposomes. Taken together, halides can be used to control interparticle distances at an extremely small scale with remarkable effects on materials properties, allowing surface probing, biosensor development, and fundamental surface science studies.

Zn2+ Induced Irreversible Aggregation, Stacking, and Leakage of Choline Phosphate Liposomes.

The interaction between lipids and metal ions is important for metal sensing, cellular signal transduction, and oxidative lipid damage. While most previous work overlooked the phosphate group of lipids for metal binding, we herein highlight its importance. Phosphocholine (PC) and its headgroup inversed choline phosphate (CP) lipids were used to prepare liposomes. From dynamic light scattering (DLS), Zn2+ causes significant aggregation or fusion of the CP liposomes, but not PC liposomes. The size change induced by Zn2+ is not fully reversed by adding EDTA, implying liposome fusion induced by Zn2+. Isothermal titration calorimetry (ITC) shows that binding between Zn2+ and CP liposomes is endothermic with a Kd of 110 µM Zn2+, suggesting an entropy driven reaction likely due to the release of bound water. In comparison, no heat was detected by titrating Zn2+ into PC liposomes or Ca2+ into CP liposomes. Furthermore, Zn2+ causes a transient leakage of the CP liposomes, and further leakage is observed upon removing Zn2+ by EDTA. Transmission electron microscopy (TEM) with negative stained samples showed multilamellar CP lipid structures attributable to Zn2+ sandwiched between lipid bilayers, leading to a proposed reaction mechanism. This work provides an interesting system for studying metal interacting with terminal phosphate groups in liposomes, affecting the size, charge, and membrane integrity of the liposomes.