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utes in dilutions of ethanol in water (70%-90%-100%), incubated in propylene oxide for 1 hour, and transferred into Epon mixed 1: 1 with propylene oxide RT overnight. Samples were moved to embedding mold filled with freshly mixed Epon, and heated for 24-48 hours at 60° C. for polymerization. Samples were analyzed on a Tecnai G2 Spirit BioTWIN electron microscope at the Elarvard Medical School EM facility.

Intravital Multiphoton Microscopy (MP-IVM) of the Popliteal LN

Naive В cells were negatively selected by magnetic isolation using CD43 beads (Miltenyi). VI10YEN В cells were labeled for 20 minutes at 37° C. with 10 iiM 5-(and 6-)-(((4-chloromethyl)benzoyl)amino)tetramethylrhod- amine (CMTMR; Invitrogen), C57BL/6 В cells were labeled for 25 minutes at 37° C. with 10 iiM 7-amino-4-chlorom- ethylcoumarin (CMAC; Invitrogen). In some experiments, labels were swapped between wildtype and VI10YEN В cells to exclude unspecific dye effects. 5-6x10^б В cells of each population were mixed and adoptively transferred by tail vein injection into C57BL/6 recipient mice one day before analysis. In some experiments, recipient C57BL/6 mice had received an injection of 30 pl CLL into the hind footpad 7-10 days before the experiment to eliminate SCS macrophages (Delemarre et al., 1990, J. Leukoc. Biol., 47: 251; incorporated herein by reference). Eighteen hours following adoptive В cell transfer, recipient mice were anaesthetized by intraperitoneal injection on of ketamine (50 mg/kg) and xylazine (10 mg/kg). The right popliteal LN was prepared microsurgically for MP-IVM and positioned on a custom-built microscope stage as described (Mempel et al., 2004, Nature, 427: 154; incorporated herein by reference). Care was taken to spare blood vessels and afferent lymph vessels. The exposed LN was submerged in normal saline and covered with a glass coverslip. A thermocouple was placed next to the LN to monitor local temperature, which was maintained at 36-38° C. MP-IVM was performed on a BioRad 2100MP system at an excitation wavelength of 800 nm, from a tunable MaiTai Ti: sapphire laser (Spectra-Phys- ics). Fluorescently labeled VSV (20 pg in 20 pl) was injected through a 31 G needle into the right hind footpad of recipient mice concomitant to observation. For four-dimensional offline analysis of cell migration, stacks of 11 optical x-y sections with 4 pM z spacing were acquired every 15 seconds with electronic zooming to 1. 8x-3x through a 20x/0. 95 water immersion objective (Olympus). Emitted fluorescence and second harmonic signals were detected through 400/40 nm, 450/80 nm, 525/50 nm, and 630/120 nm band-pass filters with non-descanned detectors to generate three-color images. Sequences of image stacks were transformed into volume-rendered, four-dimensional time-lapse movies using Volocity software (Improvision). 3D instantaneous velocities were determined by semi-automated cell tracking with Volocity and computational analysis by Matlab (Mathworks). Accumulation of cells at the SCS was determined by manual movie analysis performed by blinded observers. Every 2 minutes, the VI10YEN В cells and polyclonal В cells were counted at the SCS, in the superficial follicle (< 50 pm distance from the SCS) and the deep follicle (> 50 pm distance from the SCS), and ratios of VI10YEN/ polyclonal В cells was expressed for each compartment in the entire 30 minute movie.

Thoracic Duct Cannulation

For thoracic duct cannulation, mice received 200 pl olive oil p. o. 30 minutes prior to cannulation to facilitate visualization of the lymph vessels. Animals were then anesthetized with xylazine (10 mg/kg) and ketamine HC1 (50 mg/kg). A

polyethylene catheter (PE-10) was inserted into the right jugular vein for continuous infusion (2 ml/hour) of Ringer’s lactate (Abbott Laboratories, North Chicago, Ill. ) containing 1 U/ml heparin (American Pharmaceutical partners, Los Angeles, Calif. ). Using a dissecting microscope, the TD was exposed through a left subcostal incision. Silastic® silicon tubing (0. 012" I. D., Dow Coming, Midland, USA) was flushed with heparinised (50 U/ml) phosphate-buffered saline (DPBS, Mediatech, Herndon, Va. ), inserted into the cistema chyli through an approximately 0. 3 mm incision and fixed with isobutyl cyanoacrylate monomer (Nexaband®, Abbott Laboratories). The remaining part of the tubing was exteriorized through the posterior abdominal wall. Subsequently, the abdominal incision was closed using a 6-0 nonabsorbable running suture (Sofsilk, Tyco Healthcare Group, Norwalk, Colo. ). Following a 30 minute equilibration of lymph flow, animals were footpad injected with 108 pfu of VSV-IND and lymph samples were collected on ice for 6 hours. Lymph and organs were taken after 6 hours of thoracic duct lymph collection and plagued as described above. Lymph and organs were plagued as described above. In some experiments the draining popliteal and paraaortic lymph nodes were surgically excised and the surrounding lymph vessels cauterized to prevent lymph borne viral access to the blood.

Results and Discussion

Lymph nodes (LNs) prevent systemic dissemination of pathogens, such as viruses that enter the body’s surfaces, from peripheral sites of infection. They are also the staging ground of adaptive immune responses to pathogen-derived antigens (von Andrian and Mempel, 2003, Nat. Rev. Immunol., 3: 867; and Karrer et al, 1997, J. Exp. Med., 185: 2157; both of which are incorporated herein by reference). It is unclear how virus particles are cleared from afferent lymph and presented to cognate В cells to induce antibody responses. Here, we identify a population of CDllb* CD169⁺MHCII⁺ macrophages on the floor of the subcapsular sinus (SCS) and in the medulla of LNs that capture viral particles within minutes after subcutaneous (s. c. ) injection. SCS macrophages translocated surface-bound viral particles across the SCS floor and presented them to migrating В cells in the underlying follicles. Selective depletion of these macrophages compromised local viral retention, exacerbated viremia of the host, and impaired local В cell activation. These findings indicate that CD169⁺ macrophages have a dual physiological function. They act as innate “flypaper” by preventing the systemic spread of lymph-borne pathogens and as critical gatekeepers at the lymph-tissue interface that facilitate В cell recognition of particulate antigens and initiate humoral immune responses.

We have investigated how virus particles that enter peripheral tissues are handled within draining LNs. Hind footpads of mice were injected with fluorescently labeled UV-inactivated vesicular stomatitis virus (VSV), a cyto- pathic rhabdovirus that is transmittable by insect bites (Mead et al., 2000, Ann. N. Y. Acad. Sci., 916: 437; incorporated herein by reference) and elicits T-independent neutralizing В cell responses (Bachmann et al., 1995, Eur. J. Immunol., 25: 3445; incorporated herein by reference). Using multiphoton intravital microscopy (MP-IVM) in popliteal LNs (Mempel et al., 2004, Nature, 427: 154; incorporated herein by reference) draining the injected footpad, we observed that VSV accumulated in discrete patches on the SCS floor within minutes after sc injection, while the parenchyma and roof of the SCS remained free of virus (FIG. 11A). The viral deposits became progressively denser

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forming conspicuous irregular reticular patterns, which remained fixed in place for hours.

To characterize the predilection sites for VSV binding in LNs, we reconstituted irradiated Act(EGFP) mice with wildtype bone marrow. The resulting B6—»Act(EGFP) chimeras expressed EGFP in non-hematopoietic cells, presumably lymphatic endothelial cells, on the SCS floor and roof. Upon footpad injection of fluorescent VSV into C57BL/6—»Act (EGFP) chimeras, viral particles flooded the SCS. Three hours later, unbound lumenal VSV had disappeared, but the SCS floor displayed prominent patches of VSV that did not colocalize with EGFP⁺ cells, suggesting that VSV was captured by hematopoietic cells (FIG. 11B). To characterize the putative VSV-capturing leukocytes, we performed electron microscopy on popliteal LNs that were harvested 5 min after VSV injection (FIG. 11C). Bullet-shaped, electron- dense VSV particles were selectively bound to discrete regions on the surface of scattered large cells that resided within the SCS or just below the SCS floor. VSV-binding cells that were located beneath the SCS floor were typically in contact with the lymph compartment via protrusions that extended into the SCS lumen.

Ultrastructural studies of LNs have shown that the SCS contains many macrophages (Clark, 1962, Am. J. Anat., 110: 217; and Farr et al., 1980, Am. J. Anat., 157: 265; both of which are incorporated herein by reference), so we hypothesized that the VSV-retaining cells belonged to this population. Indeed, confocal microscopy of frozen LN sections obtained thirty minutes after footpad injection showed that VSV co-localized in the SCS with a macrophage marker, CD169/sialoadhesin (FIG. 11D). Using flow cytometry, we detected CD169 on approximately l%-2% of mononuclear cells (MNCs) in LNs, which uniformly co-expressed CDllb and MHC-II, indicating that the VSV-binding cells are indeed macrophages (FIG. 12). Most CD169⁺ cells also expressed other macrophage markers, including CD68 and F4/80, while few expressed the granulocyte/monocyte marker Gr-1. CD169⁺ cells also expressed CDllc, but at lower levels than CDllc⁷" ¹¹⁷' conventional dendritic cells (DCs). We conclude that intact virions enter the lymph within minutes after transcutaneous deposition and accumulate rapidly and selectively on macrophages in the medulla and SCS of draining LNs.

To explore mechanisms for virus fixation, live VSV (20 pg containing 2xl0⁸ pfu) was injected into hind footpads and viral titers in draining LNs were assessed 2 hours later. There was no defect in VSV retention in draining LNs of complement C3-deficient mice (FIG. 11E). DH-LMP2a mice, which lack secreted immunoglobulins, had reduced virus titers in spleen, but not in popliteal LNs (FIG. 11F). Therefore, VSV fixation in LNs occurs via a mechanism distinct from that used by splenic marginal zone macrophages, which require C3 and natural antibodies to capture blood-borne VSV (Ochsenbein et al., 1999, J. Exp. Med., 190: 1165; and Ochsenbein et al., 1999, Science, 286: 2156; both of which are incorporated herein by reference). Conceivably, the VSV surface glycoprotein (VSV-G) may be recognized in LNs by macrophage-expressed carbohydrate- binding scavenger receptors (Taylor et al., 2005, Ann. Rev. Immunol., 23: 901; incorporated herein by reference), but the precise mechanism will require further investigation.

What are the consequences of viral capture by macrophages for virus dissemination and anti-viral immunity? To address this question, we depleted LN-resident macrophages by footpad injection of clodronate liposomes (CLL; Dele- marre et al., 1990, J. Leukoc. Biol., 47: 251; incorporated herein by reference). At the dose used, sc injected CLL

selectively eliminated macrophages in LNs draining the injection site, including the popliteal, inguinal and paraortic LNs (Delemarre et al., 1990, J. Leukoc. Biol., 47: 251; incorporated herein by reference), while macrophages in distal LNs and spleen were spared (FIGS. 13 A, B). Among the different LN-resident CDllb⁺MHCII⁺ phagocytes, CLL preferentially removed the CD169⁺ subset, whereas LYVE- 1⁺ cells and conventional DCs remained unchanged. CLL- treated popliteal LNs had increased В cell numbers and enlarged follicles 7 days after treatment, but other morphological parameters, e. g. demarcation of the T/B border and SCS ultrastructure remained unaltered (FIGS. 13 C-E).

Compared to untreated LNs, we recovered approximately 10-fold lower viral titers from the draining LNs of CLL- treated mice (FIG. 11 G), suggesting that macrophage depletion rendered lymph filtration inefficient. Indeed, VSV titers were dramatically increased in blood, spleen, and nondraining LNs of CLL-treated mice. Viral dissemination from the injection site to the blood depended strictly on lymph drainage, because circulating VSV was undetectable when virus was injected into footpads of mice that carried an occluding catheter in the thoracic duct (TD), even in CLL- treated mice. Viral titers were low, but detectable in TD lymph fluid of untreated mice, but increased significantly in CLL-treated animals (FIG. 11H). This indicates that the principal conduit for early viral dissemination from peripheral tissues is the lymph, which is monitored by LN-resident, CLL -sensitive macrophages that prevent the systemic spread of lymph-borne VSV.

This capture mechanism was not unique to VSV; CD169⁺SCS macrophages also retained adenovirus (AdV; FIGS. 14 A-C) and vaccinia virus (W, FIG. 14D), indicating that macrophages act as guardians against many structurally distinct pathogens. In contrast, virus-sized latex beads (200 nm) were poorly retained in the SCS after footpad injection (FIG. 14E). Thus, SCS macrophages discriminate between lymph-borne viruses and other particles of similar size. Fluorescent VSV, AdV and W also accumulated in the medulla of draining LNs, where they were not only bound by CD169⁷'”⁴' cells (FIG. 11D) but also by CD169" LYVE-1⁺lymphatic endothelial cells (FIGS. 14 C, D). This was corroborated in CLL-treated LNs, where VSV accumulated exclusively on medullary LYVE-1⁺ cells (FIG. 15).

Next, we examined how captured VSV is recognized by В cells. Popliteal LNs contain rare В cells in the SCS lumen (FIG. 16A), but we found no evidence for virus-binding lymphocytes within the SCS on electron micrographs. Instead, viral particles were presented to В cells within superficial follicles by macrophages that extended across the SCS floor. Following injection of either VSV (FIG. 17A) or AdV (FIGS. 16 B-E), virions were readily detectable at В cell-macrophage interfaces for at least 4 hours. This suggested that SCS macrophages shuttle viral particles across the SCS floor for presentation to В cells. Transcytosis seemed unlikely, because the few vesicles containing VSV in SCS macrophages showed evidence of viral degradation. In addition, we did not detect substantial motility of virusbinding macrophages by MP-IVM, at least during the first 6 hours after challenge. Therefore, viral particles most likely reached the LN parenchyma by moving along the macrophage surface. Of note, VSV and other antigens are also presented to В cells by DCs immigrating from peripheral locations (Ludewig et al., 2000, Eur. J. Immunol., 30: 185; and Qi et al., 2006, Science, 312: 1672; both of which are incorporated herein by reference), but footpad-derived DCs are not likely to play a role during these very early events, because their migration into popliteal LNs takes much

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longer. We conclude that the SCS floor is not unsurmount- able for lymph-bome viruses; CD169⁺ macrophages appear to act as gatekeepers and facilitators of viral translocation and presentation to В cells.

Next, we explored how naive В cells respond to viral encounter using two VSV serotypes, Indiana (VSV-IND) and New Jersey (VSV-NJ) (FIG. 18; Roost et al., 1996, J. Immunol. Methods, 189: 233; incorporated herein by reference). We compared wildtype В cells to В cells from VI10YEN mice, which express a VSV-IND-specific В cell receptor that does not bind VSV-NJ (Hangartner et al., 2003, Proc. Natl. Acad. Sci., USA, 100: 12883; incorporated herein by reference). By contrast, a small fraction (2%-5%) of wildtype В cells bound both serotypes without being activated. This might reflect low-aflinity reactivity with VSV-G or indirect interactions, e. g. via complement (Rossbacher and Shlomchik, 2003, J. Exp. Med., 198: 591; incorporated herein by reference). To assess in vivo responses, differentially labeled wildtype and VI10YEN В cells were adoptively transferred and allowed to home to LN follicles. Fluorescent UV-inactivated vims was then injected into footpads and popliteal LNs were recorded by MP-IVM about 5-35 minutes later. In virus-free LNs or after injection of VSV-NJ, VI10YEN and control В cells displayed the same distribution (FIGS. 17 B-C). In contrast, upon VSV- IND injection VI10YEN cells rapidly accumulated below and at the SCS floor. There was no difference in baseline В cell motility and distribution between CLL-treated and untreated LNs, suggesting that VSV-specific В cells are equally likely to probe the SCS in both conditions. However, in CLL-treated LNs, fluorescent virus was not retained in the SCS and VI10YEN В cells failed to congregate in that region, indicating that SCS macrophages are essential for both events (FIG. 17B).

To rigorously quantify VI10YEN В cell distribution, LNs were harvested 30 minutes after VSV challenge and analyzed by confocal microscopy. While the entire follicular VI10YEN population retained its overall distribution (FIG. 17D), the subset of cells residing < 50 pm below the SCS shifted toward the SCS in VSV-IND, but not VSV-NJ containing LNs (FIG. 17E). It seems unlikely that VI10YEN В cells redistributed to the SCS because of chemoattractant signals, since unresponsive polyclonal В cells express the same chemoattractant receptors. More likely, the random contacts of motile VI10YEN cells with macrophage-bound VSV-IND triggered a BCR-dependent “stop signal” (Okada et al., 2005, PLoS Biol., 3: el50; incorporated herein by reference): Short-term exposure to VSV-IND activates LFA-1 and/or a4 integrins (Dang and Rock, 1991, J. Immunol., 146: 3273; incorporated herein by reference) on VI10YEN В cells, resulting in adhesion to the respective ligands, ICAM-1 and VCAM-1, which are both expressed in the SCS (FIG. 19). Additionally, VSV-IND bound to SCS macrophages may provide a substrate for VI10YEN В cell adhesion directly via the BCR.

To investigate how captured virions are processed upon detection by В cells, we tested В cells from VIlOYENx MHCII-EGFP mice, which allowed us to visualize endocy- tosed VSV co-localizing with endosomal МНС-II as an indicator of В cell priming (Vascotto et al., 2007, Curr., Opin., Immunol., 19: 93; incorporated herein by reference). Within 30 minutes after injection, VIlOYENxMHCII-EGFP В cells in the superficial follicle had extensively internalized VSV-IND, but not VSV-NJ particles (FIGS. 20 A, B). Virus-carrying VSV-specific В cells were infrequent, but detectable in deep follicles. These cells may have acquired virions from rare polyclonal В cells that carried VSV on

their surface, or may correspond to VI10YEN cells which failed to arrest at the SCS after acquiring VSV-IND.

While our histological findings demonstrate that intact virions are preferentially detected and acquired by В cells in the SCS and superficial follicle, MP-IVM measurements of В cell motility revealed broader antigen dissemination. After VSV-IND injection, VI10YEN cells exhibited a rapid drop in velocity throughout the entire В follicle, (FIG. 21). This was equally observed in CLL-treated and control LNs, indicating that viral antigen reached В cells independent of macrophages. This antigenic material was most likely composed of free viral protein, an inevitable by-product of natural infections. Indeed, purified supernatant of our VSV stocks induced a potent calcium flux in VI10YEN В cells (FIG. 18E). Small lymph-bome proteins are known to diffuse rapidly into follicles and activate cognate В cells (Pape et al., 2007, Immunity, 26: 491; incorporated herein by reference). Accordingly, injection of viral supernatant suppressed the motility of follicular VI10YEN В cells without inducing their accumulation at the SCS, indicating that free VSV-G was contained and active within the viral inoculum. This can explain the macrophage-independent pan-follicular effect of VSV-IND injection.

To determine the kinetics of VI10YEN В cell activation upon viral encounter, we measured common activation markers (FIG. 22). The costimulatory molecule CD86 was first up-regulated 6 hours after VSV-IND challenge. CD69 was induced more rapidly, but also on polyclonal В cells, presumably by pleiotropic IFN-a signaling (Barchet et al., 2002, J. Exp. Med., 195: 507; and Shiow et al., 2006, Nature, 440: 540; both of which are incorporated herein by reference). Surface IgM (FIGS. 20 C, D) was down-regulated as early as 30 minutes after challenge reaching a maximum within 2 h when > 70% of VI10YEN cells were

Therefore, BCR internalization provided the earliest specific readout for virus-specific В cell activation. Remarkably, VI10YEN В cells in CLL-treated LNs failed to downregu- late their BCR during the first 2 hours after subcutaneous injection of 20 pg VSV-IND (FIG. 20E), indicating that SCS macrophages are necessary for efficient early presentation of captured virions to В cells.

Primed В cells eventually solicit help from CD4⁺ T cells (Vascotto et al., 2007, Curr., Opin., Immunol., 19: 93; incorporated herein by reference) for class switch recombination and germinal center formation. To contact T cells, newly activated В cells migrate toward the T/B border (Okada et al., 2005, PLoSBiol., 3: el50; and Reifet al., Nature, 416: 94; both of which are incorporated herein by reference). This mechanism operated efficiently in macrophage-sufficient mice; most VI10YEN В cells redistributed to the T/B border within 6 h after footpad injection of as little as 40 ng VSV-IND (FIGS. 20 F, H and 23). By contrast, a 100-fold higher viral dose was needed to elicit full redistribution of VI10YEN В cells in CLL-treated mice (FIGS. 20 G, H). By 12 hours after injection, most VSV-specific cells reached the T-B border, irrespective of the injected dose. Thus, even without SCS macrophages follicular В cells are eventually activated by VSV-derived antigen, albeit less efficiently.

In conclusion, we demonstrate a dual role for CD169⁺macrophages in LNs: they capture lymph-bome viruses preventing their systemic dissemination and they guide captured virions across the SCS floor for efficient presentation and activation of follicular В cells.

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Example 2

Exemplary Lipid-Based Vaccine Nanotechnology
Architectures

Liposome Nanocarriers

In some embodiments, small liposomes (10 nm-1000 nm) are manufactured and employed to deliver, in some embodiments, one or multiple immunomodulatory agents to cells of the immune system (FIG. 3). In general, liposomes are artificially-constructed spherical lipid vesicles, whose controllable diameter from tens to thousands of nm signifies that individual liposomes comprise biocompatible compartments with volume from zeptoliters (10^-21 L) to femtoliters (10^-15L) that can be used to encapsulate and store various cargoes such as proteins, enzymes, DNA and drug molecules. Liposomes may comprise a lipid bilayer which has an amphiphilic property: both interior and exterior surfaces of the bilayer are hydrophilic, and the bilayer lumen is hydrophobic. Lipophilic molecules can spontaneously embed themselves into liposome membrane and retain their hydrophilic domains outside, and hydrophilic molecules can be chemically conjugated to the outer surface of liposome taking advantage of membrane biofunctionality.

In certain embodiments, lipids are mixed with a lipophilic immunomodulatory agent, and then formed into thin films on a solid surface. A hydrophilic immunomodulatory agent is dissolved in an aqueous solution, which is added to the lipid films to hydrolyze lipids under vortex. Liposomes with lipophilic immunomodulatory agents incorporated into the bilayer wall and hydrophilic immunomodulatory agents inside the liposome lumen are spontaneously assembled.

Nanoparticle-Stabilized Liposome Nanocarriers

In some embodiments, nanoparticle-stabilized liposomes are used to deliver one or a plurality of immunomodulatory agents to cells of the immune system (FIG. 4). When small charged nanoparticles approach the surface of liposomes carrying either opposite charge or no net charge, electrostatic or charge-dipole interaction between nanoparticles and membrane attracts the nanoparticles to stay on the membrane surface, being partially wrapped by lipid membrane. This induces local membrane bending and globule surface tension of liposomes, both of which enable tuning of membrane rigidity. This aspect is significant for vaccine delivery using liposomes to mimic viruses whose stiffness depends on the composition of other biological components within virus membrane. Moreover, adsorbed nanoparticles form a charged shell which protects liposomes against fusion, thereby enhancing liposome stability. In certain embodiments, small nanoparticles are mixed with liposomes under gentle vortex, and the nanoparticles stick to liposome surface spontaneously.

Liposome-Polymer Nanocarrier

In some embodiments, liposome-polymer nanocarriers are used to deliver one or a plurality of immunomodulatory agents to cells of the immune system (FIG. 5). Instead of keeping the liposome interior hollow, hydrophilic immunomodulatory agents may be encapsulated. FIG. 3 shows liposomes that are loaded with di-block copolymer nanoparticles to form liposome-coated polymeric nanocarriers, which have the merits of both liposomes and polymeric nanoparticles, while excluding some of their limitations. In some embodiments, the liposome shell can be used to carry lipophilic or conjugate hydrophilic immunomodulatory agents, and the polymeric core can be used to deliver hydrophobic immunomodulatory agents. In certain embodiments, pre-formulated polymeric nanoparticles (40 nm-1000

nm) are mixed with small liposomes (20 nm-100 nm) under gentle vortex to induce liposome fusion onto polymeric nanoparticle surface.

Nanoparticle-Stabilized Liposome-Polymer Nanocarriers

In some embodiments, nanoparticle-stabilized liposome- polymer nanocarriers are used to deliver one or a plurality of immunomodulatory agents (FIG. 6). By adsorbing small nanoparticles (1 nm-30 nm) to the liposome-polymer nanocarrier surface, the nanocarrier has not only the merit of both aforementioned nanoparticle-stabilized liposomes (FIG. 4) and aforementioned liposome-polymer nanoparticles (FIG. 5), but also tunable membrane rigidity and controllable liposome stability.

Liposome-Polymer Nanocarriers Comprising Reverse Micelles

In some embodiments, liposome-polymer nanocarriers containing reverse micelles are used to deliver one or a plurality of immunomodulatory agents (FIG. 7). Since the aforementioned liposome-polymer nanocarriers (FIGS. 5 and 6) are limited to carry hydrophobic immunomodulatory agents within polymeric nanoparticles, here small reverse micelles (1 nm-20 nm) are formulated to encapsulate hydrophilic immunomodulatory agents and then mixed with the di-block copolymers to formulate polymeric core of liposomes.

In certain embodiments, a hydrophilic immunomodulatory agent to be encapsulated is first incorporated into reverse micelles by mixing with naturally derived and non-toxic amphiphilic entities in a volatile, water-miscible organic solvent. The resulting biodegradable polymer-reverse micelle mixture is combined with a polymer-insoluble hydrophilic non-solvent to form nanoparticles by the rapid diffusion of the solvent into the non-solvent and evaporation of the organic solvent. Reverse micelle contained polymeric nanoparticles are mixed with lipid molecules to form the aforementioned liposome-polymer complex structure (FIG. 5).

Nanoparticle-Stabilized Liposome-Polymer Nanocarriers Comprising Reverse Micelles

In some embodiments, nanoparticle-stabilized liposome- polymer nanocarriers containing reverse micelles are used to deliver one or a plurality of immunomodulatory agents (FIG. 8). By adsorbing small nanoparticles (1 nm-30 nm) to a liposome-polymer nanocarrier surface, the nanocarrier has not only the merit of both aforementioned nanoparticle- stabilized liposomes (FIG. 4) and aforementioned reverse micelle contained liposome-polymer nanoparticles (FIG. 7), but also tunable membrane rigidity and controllable liposome stability.

Lipid Monolayer-Stabilized Polymeric Nanocarrier

In some embodiments, lipid monolayer stabilized polymeric nanocarriers are used to deliver one or a plurality of immunomodulatory agents (FIG. 9). As compared to aforementioned liposome-polymer nanocarrier (FIGS. 5-8), this system has the merit of simplicity in terms to both agents and manufacturing. In some embodiments, a hydrophobic homopolymer can form the polymeric core in contrast to the di-block copolymer used in FIGS. 5-8, which has both hydrophobic and hydrophilic segments. Lipid-stabilized polymeric nanocarriers can be formed within one single step instead of formulating polymeric nanoparticle and liposome separately followed by fusing them together.

In certain embodiments, a hydrophilic immunomodulatory molecule is first chemically conjugated to a lipid headgroup. The conjugate is mixed with a certain ratio of unconjugated lipid molecules in an aqueous solution containing one or more water-miscible solvents. A biodegrad

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