Polysorbasome: a Colloidal Vesicle Contoured by Polymeric

Subscriber access provided by UNIVERSITY OF TOLEDO LIBRARIES

Biological and Medical Applications of Materials and Interfaces

Polysorbasome: a Colloidal Vesicle Contoured by Polymeric Bioresorbable Amphiphiles as an Immunogenic Depot for Vaccine Delivery Chiung-Yi Huang, Chung-Hsiung Huang, Shih-Jen Liu, HsinWei Chen, Chih-Hsiang Leng, Pele Chong, and Ming-Hsi Huang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b03044 • Publication Date (Web): 29 Mar 2018 Downloaded from http://pubs.acs.org on March 29, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Polysorbasome: a Colloidal Vesicle Contoured by Polymeric Bioresorbable Amphiphiles as an Immunogenic Depot for Vaccine Delivery Chiung-Yi Huang,† Chung-Hsiung Huang,† Shih-Jen Liu,†,‡ Hsin-Wei Chen,†,‡ Chih-Hsiang Leng,†,‡ Pele Chong,† and Ming-Hsi Huang*,†,‡

National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes,

35053 Miaoli, Taiwan

Graduate Institute of Biomedical Sciences, China Medical University, 40402 Taichung, Taiwan

ACS Paragon Plus Environment

1

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 31

ABSTRACT: To accomplish innovative vaccine design, two key challenges are related to developing formulations that avoid cold chain shipment and finding a delivery vehicle that is absorbable in vivo. Here we explored the design and performance of a colloidal vesicle that enabled us to consider both challenges. We used polymeric bioresorbable amphiphiles as surface-active agents for stabilizing oily/aqueous interfaces and formed a colloidal vehicle named polysorbasome (polymeric absorbable vesicle), without using conventional emulsifiers such as sorbitan esters or their ethoxylates. Homogenizing the oil/water compartments forms a colloid containing an aqueous solution in its core and provides an oily barrier that isolates the encapsulated material from external materials. In this form, the polysorbasome serves as a depot for sustained delivery of vaccine antigens. Following vaccination, the antigen-specific antibodies and the cell-mediated immunity can be manipulated after the antigen being formulated with polysorbasome particles. Then, the degradability intrinsic to the polymeric bioresorbable amphiphiles complies with the destruction and further absorbance of the vehicles in vivo. The structural features of these versatile vesicles based on bioresorbable amphiphilic engineering may provide new insights into vaccine delivery.

KEYWORDS: bioresorbable amphiphiles, colloids, emulsions, polysorbasomes, vaccine adjuvants


2

Page 3 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION An emulsion is a quasi-stable colloidal dispersion which encompasses at least two immiscible liquids. Usually, water and biocompatible/metabolizable oils are the two practical components in pharmaceutical and vaccine formulations.1,2 Emulsions are stabilized by adding appropriate surface-active agents called emulsifiers that localize at the oily/aqueous interfaces, where they reduce the surface free energy.2,3 The hydrophilic-lipophilic balance (HLB) of the emulsifier plays a governing role in the colloid dispersion.3-5 The water-in-oil (W/O) dispersion is prepared by a lipophilic emulsifier, such as those derived from sorbitan fatty acid esters (known as SPAN®) or poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (known as Pluronics).6 Conversely, the oil-in-water (O/W) dispersion is elaborated by a hydrophilic emulsifier, such as those derived from ethoxylated sorbitan fatty acid esters (known as TWEEN®).1,2 As with any emulsion that deals with double compartment structure (W/O/W), combinations of multiple emulsifiers can be used to tune the emulsifying system to an intermediate HLB value.3-5 Generally, vaccines contain sensitive biological ingredients that suffer damage and reduce their potency when exposed to suboptimal temperatures.7 Cold chain refrigeration is necessary to preserve the immunogenicity of vaccine antigens for long-term storage of most vaccines;7 however, this represents an important economic impact, especially for communities that lack cold chain facilities. In most cases, attempts have focused on the feasibility of vaccine shipment in the solid dosage form (lyophilized powder), even in extreme climates, following on-site reconstitution by adding the liquid diluents to vaccine powder before administration;8 yet, the freeze-drying process might be very delicate to carry out when the vaccine formulation consists of an emulsion that may undergo fracturing rather than plastic deformation. Another concern is


3


Page 4 of 31

that medical risks, such as safety and tolerability of the subject, are addressed in the case of prophylactic vaccines because they are often employed in populations of infants and young children.9,10 Emulsion-based adjuvants were demonstrated as an effective tool to increase vaccine efficacy;1,3 nevertheless, it has been well documented that the emulsions made by the aforementioned emulsifiers may either meet the requirements of stability at suboptimal temperature7 or cause serious side effects, including severe non-immunological anaphylactoid reactions.11 Therefore, there is an unmet medical need to increase the number of well-defined emulsifiers in the preparation of emulsion-based vaccine adjuvants towards enhancing the vaccine efficacy without alternating the safety aspects. In this report, we describe the use of polymeric bioresorbable amphiphiles (PEGylated polyesters/sorbitan polyesters) as emulsifying agents to stabilize oily/aqueous interfaces and give rise to oil-shelled colloids (Figure 1), without using conventional emulsifiers such as sorbitan esters or their ethoxylates. In this form, the colloids contain an aqueous solution in their core and provide an oily barrier that isolates the encapsulated material from external materials. Following vaccination, the colloidal vesicles can act as a depot for sustained release of vaccine antigens. Then, random chain scission of the lipophilic moiety of the main chain polymer progressively changes the amphiphilic behaviors intrinsic to emulsifiers, thus affecting the stability of the colloids.

MATERIALS AND METHODS Study design. Polymeric bioresorbable amphiphiles consist of hydrophilic groups made from PEG and sorbitan and, lipophilic groups comprised polyesters derived from lactide and ε-


4

Page 5 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


caprolactone. Elaboration of polysorbasomes was performed in a similar way to the emulsion. A stability study was conducted at temperatures of 4°C, 25°C and 37°C, which mimic cold chain, ambient and extreme storage conditions, respectively. The temperature of 37°C was also selected to mimic the post-administration stage of vaccine formulation. The impact of the immunoavailability of antigens on the production of antibodies was investigated in a mouse model with ovalbumin as the antigen and was executed with six animals per group. Pooled data from three mice are shown for the draining LN cells and T-cell immunity experiments to obtain sufficient replicates for each condition. Wild-type female BALB/c mice (in vivo imaging and humoral immunoassays) and C57BL/6 mice (cellular immunoassays), aged 5 weeks, were obtained from the National Laboratory Animal Centre and housed at the Laboratory Animal Centre (LAC) of the NHRI, which has received accreditation from AAALAC. The number of animals needed was based on the minimum number of mice required to perform immune response analysis, in addition to the numbers needed for statistical analysis and replicate experiments. Before treatment, animals were randomized to minimize variances between groups. All work on animals was performed under an IACUC-approved protocol (NHRI-IACUC-103027-AC) in accordance with the guidelines of the LAC of NHRI. All experiments were performed independently at least twice. Materials. Polyethylene glycol 5000 monomethyl ether (MePEG5000) was purchased from Fluka (Buchs, Switzerland). ε-Caprolactone was obtained from Acros Organics (Geel, Belgium). Stannous octoate (SnOct2), phosphate-buffered saline (PBS), D-sorbitol, dimethyl sulfoxide-d6 (DMSO-d6), ovalbumin (OVA, grade V) and squalene were supplied from Sigma (St. Louis, MO, USA). These materials were used as received. DL-Lactide (3,6-dimethyl-1,4-dioxane-2,5-


5


Page 6 of 31

dione) was obtained from Aldrich (Seelze, Germany) and purified by recrystallization from acetone prior to use. All solvents were of analytical grade. Measurements. Two-dimensional diffusion ordered spectroscopy (2D DOSY) NMR measurements were performed at 300 K on a 600-MHz Varian VNMRS-600 NMR spectrometer (Varian, Palo Alto, CA, USA), using DMSO-d6 as solvent. Chemical shifts were shown in ppm with tetramethylsilane as the reference. Size exclusion chromatography (SEC) was carried out on an apparatus equipped with an HPLC pump (LabAllianceTM series III, Scientific System Inc., USA), a refractive index (RI) detector (LabAllianceTM RI-101, Scientific System Inc., USA), two size exclusion columns (Agilent Technologies, Inc., UK) connected in series, one guard column (PLgel 5-µm, 7.5 × 50 mm) and one mixed-D column (PLgel 5-µm, 7.5 × 300 mm), the mobile phase being tetrahydrofuran (THF) and the flow rate of 1.0 ml/min. The number-average molecular weight (Mn) and polydispersity index (Đ) of the polymer were expressed relative to a calibration curve made with polystyrene standards (Varian, Inc., Amherst, MA, USA). Mass spectrometry measurements were recorded using a Micromass® MALDI micro MXTM time-offlight mass spectrometer (Waters®, Milford, MA, USA). Polymer samples were prepared from 0.2% trifluoroacetic acid in a binary mixture of acetonitrile/water (1:1, v/v), using α-acyano-4hydroxy cinnamic acid (CHCA, Sigma, Steinheim, Germany) as a matrix with sodium trifluoroacetate (Na-TFA; Fluka, Buchs, Switzerland). To monitor the low-molecular-weight components, the samples were deposited without the matrix onto a plate containing porous silicone spots (Waters® MassPREPTM DIOS-targetTM plate, Milford, MA, USA). Preparation of a PEGylated polyester. Poly(ethylene glycol)-block-poly(lactide-co-εcaprolactone) (PEG-PLACL) was made by bulk ring-opening polymerization of lactide and εcaprolactone onto a monomethoxy PEG, as previously described.12


6

Page 7 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


First, predetermined amounts of MePEG5000 (43 g), lactide (12 g), ε-caprolactone (9.5 g) and SnOct2 (0.5 wt %) were placed into a polymerization ampoule. After degassing at 140°C, the ampoule was first cooled down to room temperature, sealed in vacuo, and was allowed to rotate gently at 140°C for 24 h. The copolymer was dissolved in acetone and then reprecipitated from ethanol. The recovered polymer was finally collected by filtration to yield the pure product and dried under reduced pressure. The compositional and molecular characteristics of the resulting polymer were characterized by 1H NMR and SEC. The [LA]/[CL]/[OE] molar ratio of 0.078/0.045/1 was determined from the integrations of the bands at 3.6 ppm (for PEG), at 4.1 ppm (for PCL), and at 5.2 ppm (for PLA) in the 1H NMR spectra. The Mn of the polymer and the weight ratio of the hydrophilic portion to the lipophilic moiety were calculated according to the following equation: Mn(NMR, DMSO-d6) = Mn,PEG + Mn,PLACL = 5,000 + (72×5,000/44×[LA]/[OE] + 114×5,000/44×[CL]/[OE]) = 6,700 (g/mol) WPEG:WPLACL = Mn,PEG:Mn,PLACL = 75% : 25%. where 5,000 is the average molecular weight of PEG indicated by the supplier; and 44, 72 and 114 correspond to the molecular weights of oxyethylene (OE), lactyl (LA), and caproyl (CL) repeat units, respectively. Mn(SEC) of 7,000 g/mol and Đ of 1.1 were monitored by SEC, with the data being expressed with respect to polystyrene standards.


7


Page 8 of 31

Preparation of a sorbitan-polyester. First, a predetermined amount of sorbitol was added to the flask. Sorbitan was prepared simply by distilling water out of sorbitol at 180°C for 2 h in the presence of phosphoric acid (J.T. Baker®, PA, USA) using a Rotavapor® R-210 (BÜCHI Labortechnik AG, Switzerland) under vacuum.13 Sorbitan-polyester was synthesized in the same manner as PEG-PLACL. Briefly, predetermined amounts of sorbitan, lactide, ε-caprolactone, and SnOct2 (0.5 wt %) were placed into a polymerization ampoule. After degassing at 140°C, the ampoule was first cooled down to room temperature, sealed in vacuo, and was allowed to rotate gently at 140°C for 24 h. The resulting sorbitan-poly(lactide-co-ε-caprolactone) copolymer (named sorbitan-PLACL) was extracted with 100 ml of dichloromethane, and then washed with 50 ml of distilled water. The solvent of the organic phase was partially evaporated under reduced pressure. Polysorbasome (PSS) preparation. In a typical procedure, 120 mg of PEG-PLACL, 200 mg of sorbitan-PLACL, 0.76 ml of antigen medium (a particular concentration of OVA diluted in PBS) and 0.92 ml of squalene oil were homogenized and assembled by a homogenizer (Polytron® PT 2500, Kinematica AG, Switzerland) under 6,000 rpm for 5 min. The resulting colloidal formulation served as a stock for further applications. The microscopic aspects of the colloids were monitored by an optical microscope combined with Olympus DP70 digital camera system. The particle size was measured in a Brookhaven 90 plus particle size analyzer (Brookhaven Instruments LTD., NY, USA). In vitro release studies were conducted by the inverted dialysis tube method, as described previously.3 Briefly, OVA-loaded formulations (3 mg per 0.3 ml) were added in the sample reservoir of a dialysis chamber (MWCO: 0.2 µm). The chamber was then immersed inversely in a 50-ml centrifugal tube, and 2 ml of PBS was supplemented. The tubes were tightly sealed and


8

Page 9 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


allowed to place in a 37°C circulating water bath. At designated intervals, 100 µl of sample was taken with a pipette from the release medium exterior to the reservoir, and replaced with the same amount of fresh PBS buffer. The OVA release assays were carried out by the bicinchoninic acid method according to the supplier's instructions (BCATM Protein Assay Kit, Pierce, Rockford, IL, USA). Hydrolytic degradation. 20 µl of stock polysorbasomes were dispersed in an Eppendorf tube filled with 80 µl of PBS solution. The tubes were allowed to place in a circulating water bath at 37°C. Three specimens were collected at predetermined time points, followed by lyophilization before being subjected to SEC analysis. Nanosorbasome preparation. To prepare polysorbasome at the nanoscale, colloidal suspensions were investigated by redispersing 200 µl of the PSS stock in 1,800 µl of PBS solution and mixing the suspension with a test-tube rotator at 5 rpm for 1 h. Samples with the final concentration of 1/1,000 v/v in PBS were extruded successively through 1.0-µm, 0.4-µm, 0.2-µm, and 0.1-µm polycarbonate filters (Whatman®NucleporeTM Track-Etched Membranes, GE Healthcare Companies, Kent, UK) equipped in a mini-extruder fitted with two 1.0-ml Hamilton syringes. The samples were subjected to 11 passes through each extruder membrane following the supplier's instructions.14 In vivo fluorescence imaging and histological examination. The distribution of antigen in vivo was measured by tracking ovalbumin protein conjugated with Alexa Fluor® 647 (OVA*, Ex: 652 nm, Em: 668 nm, Molecular Probes, Eugene, OR, USA) after dispersing the formulations in PBS. BALB/c mice (n = 3 per group) were injected in both quadriceps with 50 µl per quadriceps of OVA* (5 µg) diluted in PBS (control group) or formulated with 10% v/v PSS


9


Page 10 of 31

(stabilized by PEG-PLACL/sorbitan-PLACL 1:4:4). The animals were anaesthetized by vapor isoflurane using the XGI-8 anesthesia system. At 0, 24, and 72 h post-injection, the fluorescence image was recorded using an IVIS optical imaging system (Xenogen IVIS® Spectrum 200, Caliper Life Sciences, USA). The intensity of fluorescent signals was acquired and analyzed using the IVIS Living Image 4.0 software package. For histological examination, mice were sacrificed on weeks 1, 3 and 7 post-injection. The muscles at the injection sites were excised, sectioned, and stained with hematoxylin and eosin (H&E) by the Pathology Core Laboratory of NHRI. Humoural immunoassay. To determine the antigen-specific IgG titers, BALB/c mice (n = 6 per group) were injected once intramuscularly with 10 µg of OVA, either in 100 µl of sterilized PBS or formulated with 10% v/v PSS (stabilized by PEG-PLACL/sorbitan-PLACL 1:4:4). Serum samples were collected from the submandibular vein of the vaccinated mice, followed by clotted and centrifuged, and were heat inactivated at 56°C for 30 min. The presence of anti-OVA IgG in the sera was assayed by enzyme-linked immunosorbent assay (ELISA), as described previously.15 The titers were read as the maximum dilution that resulted in a 450 nm OD reading at least two times more than that of the control samples (pre-immune sera). An undetectable level was recorded as a titer equal to 500. A comparison between the antibody titers of the polysorbasome-adjuvant group and those of the no adjuvant group was made using the two-tailed Student's t-test on log10-transformed IgG titers, using Microsoft Excel. Analysis of lymphoid APC activation and T-cell immunity. To examined cell-mediated immunity, C57BL/6 mice (n = 3 per group) were injected subcutaneously in both hindlimbs with 50 µl per hindlimb of OVA (total 10 µg per mouse in 100 µl of sterilized PBS) or formulated


10

Page 11 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


with PSS (stabilized by PEG-PLACL/sorbitan-PLACL 1:4:4, 10% v/v PSS of the total volume) or an O/W-type emulsion (AddaVaxTM, a squalene-based oil-in-water emulsion; InvivoGen). Lymphoid APC activation was evaluated three days after vaccination, using the draining lymphoid cells collected from the inguinal and popliteal lymph nodes of the vaccinated mice, as described previously.15 The red blood cells were depleted at ambient temperature for 1 min in ACK-lysing buffer containing 155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA. The cell pellets were harvested, washed with FACS buffer and stained with FITC-conjugated CD11c, PEconjugated CD40, PeCy7-conjugated CD86 and Alexa 647-conjugated MHC II antibodies, and acquired on a LSRII flow cytometer (BD Immunocytometry Systems, CA, USA). T-cell immunity was evaluated seven days after vaccination. The splenocytes (5×106/ml) harvested from the vaccinated mice were cultured in triplicate in the presence of OVA (10 µg/ml) for 72 h. Cells incubated with medium only were used as a negative control. At 24 h, the cell pellet was washed with TRI reagent (Sigma) for total RNA extraction following the manufacturer's instructions. The expression levels of transcriptional factors T-bet, RORγt, and βactin (the reference standard) in OVA-restimulated splenocytes were measured by reverse transcription-polymerase chain reaction (RT-PCR), which were electrophoresed in 2% agarose gels and stained with 0.1 µg/ml SYBR® Green (Thermo Fisher Scientific, Inc., CA, USA). At 72 h, the supernatants were collected from triplicate culture for the measurements of cytokines IL-2, IL-17 and IFN-γ using ELISA paired antibodies following the supplier's instructions (DuoSet® ELISA Development Kit, R&D Systems, Inc., MN USA).


11


Page 12 of 31

The data are expressed as the mean ± s.d. for each treatment group. Statistical differences between the treated groups and the control group were compared by performing a two-tailed Student's t-test, using Microsoft Excel. Significance was set at p < 0.01.

RESULTS AND DISCUSSION Preparation of polymeric bioresorbable amphiphiles. The hydrophilic emulsifier used here is an AB-type diblock copolymer PEG-PLACL composed of 75 wt-% PEG as the hydrophilic block and 25 wt-% PLACL as the lipophilic block, with a calculated HLB value of 15. A series of sorbitan-PLACL copolymers were prepared from PLACL random copolymers of well-defined length that were end-capped with a sorbitan group to initiate the tin(II)-catalysed polymerization reaction (Figure 2 and Figure 3). Here, "sorbitan-PLACL 1:4:4" means that the weight ratio of sorbitan:LA:CL is 1:4:4. It is known that degradable aliphatic polyesters derived from LA enantiomers and ε-CL are considered profitable in the field of sustained delivery.9 Synthetic scheme and chemical structure of sorbitan-polyester copolymers are shown in Figure 2. Sorbitan was firstly prepared by anhydrization of sorbitol in the presence of phosphoric acid, the latter acts as a catalyst to convert sorbitol to sorbitan with high yield.13 Then, lactide and εcaprolactone were allowed to polymerize onto sorbitan molecule, resulting in a sorbitan-PLACL copolymer composed of a hydrophilic sorbitan moiety and lipophilic PLACL segments. Figure 3a shows the DOSY map of a sorbitan-PLACL copolymer in DMSO-d6. The NMR spectrum exhibited signals corresponding to the sorbitan moiety (δ = 3.9 and 4.3 ppm),16 PLA block (δ = 1.3 and 5.1 ppm),12 and PCL block (δ = 1.2, 1.5, 2.5, and 4.0 ppm).12 These signals had the same


12

Page 13 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


diffusion coefficient, indicating efficient ligation of sorbitan to the copolymer. The molecular hydrodynamic volume of the sorbitan-PLACL copolymer was presented in Figure 3b. As expected, sorbitan-PLACL exhibited a single-peak molecular weight (MW) distribution (polydispersity index of 1.4), indicating the absence of unreacted lactide and ε-caprolactone. The precise MW of the sorbitan-PLACL copolymer was measured by MALDI-TOF mass spectrometry. As shown in Figure 3c, the MW of sorbitan-PLACL was detected in the range of 300 to 1,800 g/mol. The mass spectra were well resolved with the peaks separated by 114 and 72 mass units, which corresponded to the MW of the caproyl unit and lactyl motif, respectively. Colloidal vesicles contoured by polymeric bioresorbable amphiphiles. A series of polymeric absorbable colloids were prepared by homogenizing a mixture of water-immiscible oil and an aqueous solution, the latter consisting of PEG-PLACL and sorbitan-PLACL in PBS. Initially, phase separation occurred between the oil and aqueous solution. As soon as the agitator started, the oil/water dispersion can be assembled into an isotropic colloid (Figure S1, Supporting Information). Squalene was selected as the oil component because of its biocompatibility and adjuvanticity. It is important to note that squalene alone is not an adjuvant, but emulsions of squalene are formulated into vaccines to boost the immunogenicity of antigens to enhance vaccine efficacy.1 In addition to its use as an excipient (adjuvant) in vaccine preparedness, squalene has been widely used as a skin moisturizer in cosmetics.3 In fact, squalene is the precursor of cholesterol and steroid hormones and can be easily metabolized and excreted.1 We thus designate the colloidal vesicle contoured by polymeric bioresorbable amphiphiles as a polysorbasome (abbreviated PSS) because the intention of emulsion is to stabilize oily/aqueous interfaces whereas the pursuit of a polysorbasome is to ensure the


13


Page 14 of 31

bioresorbability of a colloid, i.e., all the applied excipients show main chain cleavage into small molecules and are further absorbed in vivo. The colloidal dispersions of the so-obtained PSS can be identified by the in vitro release and droplet test. An in vitro release study was conducted using hydrophilic ovalbumin (OVA) protein as a model antigen. As seen in Figure 4a, free OVA was quickly released into the PBS aqueous solution outside of the dialysis chamber. The release of OVA from the designed PSS followed first-order Fickian diffusive release,17 i.e., the OVA trapped within the PSS first diffused from the inner core to the surface and was then released into the PBS solution, following the intermediate controlled-release mechanisms. By varying the HLB of an emulsifying system, the sustained delivery ability can easily be manipulated; sorbitan-PLACL 1:4:4- and sorbitan-PLACL 1:8:8-based PSSs presented good depot effects such that the hydrophilic OVA was slowly released over 300 h. However, the sorbitan-PLACL 1:12:12containing PSS had similar release profiles to free OVA, which could be ascribed to an emulsifier possessing an extremely low HLB value failing to homogenize the oily/aqueous interfaces. PSS possesses a high affinity for water, and an optical micrograph image shows that the droplet of PSS diffused in the water and then the colloidal clusters were dispersed into homogeneous fine particles (Figure S1, Supporting Information). Such oil-based vesicles are soft and deformable: their size can be controlled by passage through an extruder membrane. As shown in Figure 4b, the PSS suspensions initially formed large vehicles following homogenization (~1,000 nm). After successively passing through extrusion membrane filters with pore sizes of 1.0 µm, 0.4 µm, 0.2 µm, and 0.1 µm, a unimodal distribution of particles was observed with an average diameter about 100 nm. However, the number counts of the filtrates


14

Page 15 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


were reduced after being passed through pore diameters less than 0.2 µm (Figure S2, Supporting Information). It is clear that the size matter of the particulate vaccine adjuvants is the major concern regarding antigen uptake and adjuvant activities. Particles with sizes less than 100 nm are internalized by APCs through macropinocytosis, whereas O/W emulsions at the submicron scale can be internalized by APCs through phagocytosis, without specific recognition.18 Data from the in vitro release study and particle size analysis revealed that the squalene/water interfaces stabilized by an emulsifying system containing bioresorbable amphiphiles (PEGPLACL/sorbitan-PLACL) with an intermediate HLB value resulted in oil-shelled architectures consisting of both W/O and O/W compartments, i.e., the W/O dispersed in the suspending aqueous solution as the oil globules containing smaller aqueous droplets (see Supporting Information: Polysorbasome preparation). Principally, molecular weight (MW) changes reflect the hydrolytic cleavage of degradable polyesters.12 We next investigated the hydrolytic degradation characteristics of PEG-PLACL and sorbitan-PLACL within PSS, followed by size exclusion chromatography (SEC) analysis (Figure 4c). Upon suspension in buffered saline solution at 37°C, the SEC traces of PEG-PLACL and sorbitan-PLACL shifted to lower MW, in agreement with the chain scission of PLACL. As degradation proceeded, the peaks of low-MW species increased between week 2 and week 4, revealing that the majority of PLACL oligomers broke away from PEG-PLACL and sorbitanPLACL. In addition, ester bond cleavage of PLACL oligomers occurred continuously to result in further degradation, in agreement with our previous study, which showed that polymeric amphiphiles started to lose PLACL (the lipophilic moiety) after 9 weeks.15 Since the squalene/water interfaces of PSS were contoured by PEG-PLACL and sorbitanPLACL, we next investigated how the degradation of these polymeric amphiphiles affects the


15


Page 16 of 31

stability of the corresponding PSS. Figure 4d shows the stability of PSS stored at various temperatures, 4°C, 25°C and 37°C. Overall, this emulsion was stable during storage at 4°C or 25°C for the 26-week observation period. Approximately 5% of the water was observed to be disassociated at the bottom of the vial after 18 weeks, assigned to the creaming phenomenon attributed to the difference in density of the phases. In this case, the colloid could be easily reformed by simple vortex mixing. When stored at 37°C, 30% of the free oil dissociated from the colloid by week 24. Once the phase separation occurred, the colloid could no longer be reformed by simple vortex mixing. At week 26, PSS had completely disintegrated into two layers, with the lighter (oily phase) at the top and the heavier (aqueous solution) on the bottom. This feature indicates that missing the lipophilic PLACL moiety in the PEG-PLACL/sorbitan-PLACL emulsifiers directly affected the stability of PSS, leading to disintegration of the colloid and separation of the oil and water phases. The stability characteristics of a PSS are distinguished from those of an emulsion (Figure S3, Supporting Information). Principally, the physical disintegration of an emulsion is accomplished mainly by two processes: coalescence and Ostwald ripening;19 however, an assumption was proposed that the emulsifiers within the emulsion are unchanged. In the present study, we postulated that a colloid with an oily/aqueous interface stabilized by PEG-PLACL/sorbitan-PLACL not only can provide high stability to vaccine antigens carried by colloidal particles during preparation and storage but also can afford high degradability in vivo. Overall, polymeric bioresorbable amphiphiles work as surface-active agents to stabilize the oily/aqueous interfaces and yield oil-shelled colloids (polymeric bioresorbable vehicles) during preparation and storage. The degradability intrinsic to these amphiphiles complies with the vehicles' progressive destruction in vivo. These technical features


16

Page 17 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


are significant and important to vaccine development, as colloidal vehicles possessing designable stability and degradability at 37°C are important for vaccine efficacy and safety. Polysorbasomes as an immunogenic depot for vaccine antigens. Here, polysorbasomes were designed mainly for vaccine delivery. We hypothesized that the vaccine antigens could be entrapped within the designed vehicle, and then delivered from the colloidal matrix in a desired profile in vivo; hence, a single injection can achieve a prime-boost vaccination. To determine whether PSS can function as an antigen depot in vivo, mice were vaccinated intramuscularly with ovalbumin protein conjugated with Alexa Fluor®647 (abbreviated as OVA*), either alone or formulated with PSS particles. IVIS fluorescence images showed that OVA* signals at the injection sites dropped dramatically within 24 h due to OVA* degradation (Figure 5a). As expected, the signals were gradually decreased in the presence of the PSS particles, in agreement with the findings from in vitro release study. These results suggest that the PSS is a good candidate depot or carrier for stepwise release of the hydrophilic protein antigen. We next examined whether combining the targeted and prolonged releases of protein antigen could drive the production of antigen-specific antibodies. Following a single injection, mice vaccinated with OVA alone could induce a low serum antigen-specific IgG antibody titer (Figure 5b). Interestingly, antibody titers were enhanced significantly when the protein antigen was encapsulated into PSS colloidal particles. The elicited antigen-specific IgG titer was 18,000 at week 8 (20 times more than the non-adjuvant group) and lasted for at least 24 weeks, indicating an improvement in the magnitude and prolonged existence of the induced antibody responses when the antigen was formulated within the PSS particles. Histological examination of the immunized mice was carried out to monitor the biocompatibility of PSS particles (Figure 5c). A low-magnification image (x40) shows that the vacuoles (identified as PSS particles) decreased in


17


Page 18 of 31

size over time, showing the absorbance of PSS in vivo. A high-magnification image (x400) shows that the cells infiltrated all around the PSS particles, indicating the recruitment of cells at the injection sites within one week and retention for up to 7 weeks post-injection. Notably, cell necrosis and calcification were rarely visible around the injected mass. The data from the stability aspects and histological examination reveal that the PSS colloidal particles are not destroyed into two phases until internalization by immune cells, a feature that must be confronted to the requirements for vaccine adjuvant. It is well known that the induction of cell-mediated immunity is not only a critical parameter in effective vaccination by clearance of virus-infected cells,18 but is beneficial to successful cancer immunotherapy upon destruction of tumor cells.20,21 Following administration, antigen-loaded APCs in peripheral tissues migrate to local draining lymph nodes (LNs), where they fragment the antigen into antigenic peptides for presentation to lymphocytes mainly on the major histocompatibility complex (MHC) and costimulatory molecules of the cell surface.21,22 To investigate the impact of polysorbasome compounds on the APC activation, we vaccinated mice with OVA, alone or formulated with a PSS or an O/W (a TWEEN®/SPAN®-stabilized squalenein-water emulsion), followed by measuring the level of activation marker expression on CD11c+ cells harvested from the draining LNs. As shown in Figure 6a, vaccination of PSS- or O/Wformulated OVA enhanced the high expression of MHC class II and costimulatory signals CD40 and CD86 compared with OVA alone. This finding reveals that the squalene-core PSS retains the ability of the O/W to induce phenotypic changes in the draining LNs. We further investigated whether PSS-induced cell activation can help APCs present the antigens to T cells and elicit the differentiation and modulation of T cell populations, including CD4+ helper T (TH) cells. Seven days after vaccination, single-cell suspensions were harvested from the mouse spleen and


18

Page 19 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


restimulated in vitro with OVA antigen. As shown in Figure 6b, the concentrations of IFN-γ, IL2, and IL-17 in the O/W or PSS-formulated group were significantly higher than those in the non-formulated group. Consistently, elevated mRNA expression of T-bet (TH1) and RORγt (TH17) was observed in mice vaccinated with OVA formulated with PSS or O/W compared to mice vaccinated with OVA alone (Figure 6c). With similar functions to squalene-cored O/W, PSS has intermediate mechanisms between sustained-release depots and immunostimulatory activities in terms of the magnitude of the induction of antibodies and the activation of draining LNs and T cells. Finally, no obvious clinical signs of autoimmune or allergic disorders were observed in the mice following PSS injection. The presence of PSS colloids in the vaccine component substantially enhances the vaccine efficacy with conserving the safety aspects of free-antigen vaccination. Polysorbasomes or PSS, of the general type examined here, possess quasi-stable architectures consisting of the two immiscible liquids squalene/water and PEGylated polyester/sorbitan polyester amphiphiles, which form oil-shelled colloids in water in a different way from an emulsion. These polymeric absorbable vehicles have several versatile advantages over common vaccine formulations. Soft, liquid-liquid colloidal vesicles are deformable and easily injected by syringe with a small gauge needle, thereby potentially diminishing local reactions at the injection site. In addition, high softness of materials allows pliability and mobility of loaded antigens to increase the adjuvant-cell contact area.23 These features encourage the use of such vesicles for massive vaccination. Small, it is clear that the size of the particulate vaccine adjuvants is an important factor in antigen uptake as well as the adjuvant activities.18 Homogeneous colloidal particles can be controlled by passing them through an extruder membrane, and their size can therefore be controlled from 1,000 nm to 100 nm, thus facilitating


19


Page 20 of 31

the induction of appropriate immunity.24 Smart, oil-shelled capsules are useful for encapsulating and protecting designed bioactive molecules; the loss of the lipophilic moiety of amphiphiles directly affected the stability of the colloid after vaccination. Safe, all excipients used here showed main chain cleavage into small molecules and further absorbance in vivo, thereby conceptually conserving the innocuity of the vaccine. Stimulatory, the first investigation using OVA as a model antigen indicated that PSS particles are immunogenic and facilitate the activation of immune cells, such that antigen-specific antibodies and cell-mediated immunity could be manipulated after the antigen was formulated with PSS. Simple, the raw materials for PSS elaboration are commercially available and are largely used for vaccine/drug delivery; in addition, no complicating processes or supplemental equipment are required when performing the vaccine formulation, thus effectively reducing the cost.

CONCLUSIONS Stability and bioresorbability are regarded as crucial in successful vaccine delivery. Here we explore the design and performance of a colloidal vesicle that enabled us to consider both targets. We demonstrated that colloidal vesicles contoured by polymeric bioresorbable amphiphiles allow the vesicles to act as an immunogenic depot for enhancing vaccine efficacy without altering the safety aspects compared to free-antigen vaccination. These features strongly support the use of PSS for sustained delivery of bioactive agents, in particular vaccine antigens. ASSOCIATED CONTENT Supporting Information. The Supporting information related to polysorbasome preparation and stability. This material is available free of charge via the Internet at http://pubs.acs.org.


20

Page 21 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


AUTHOR INFORMATION Corresponding Author *

[email protected]

ORCID Ming-Hsi Huang: 0000-0002-9670-3821 Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Health Research Institutes of Taiwan (106A1-IVPP23014) and by a grant from the Ministry of Science and Technology of Taiwan (MOST 104-2314B-400-004-MY2). REFERENCES (1) Vogel, F. R.; Caillet, C.; Kusters, I. C.; Haensler, J. Emulsion-based Adjuvants for Influenza Vaccines. Expert Rev. Vaccines 2009, 8, 483–492. (2) Strickley, R. G. Solubilizing Excipients in Oral and Injectable Formulations. Pharm. Res. 2004, 21, 201–230. (3) Aucouturier, J.; Dupuis, L.; Ganne, V. Adjuvants Designed for Veterinary and Human Vaccines. Vaccine 2001, 19, 2666–2672. (4) Griffin, W. C. Classification of Surface-Active Agents by “HLB”. J. Soc. Cosmet. Chem. 1949, 1, 311–326. (5) Griffin, W. C. Calculation of HLB Values of Non-Ionic Surfactants. J. Soc. Cosmet. Chem. 1949, 1, 249–256.


21


Page 22 of 31

(6) Leenaars, M.; Koedam, M. A.; Hendriksen, C. F.; Claassen, E. Immune Responses and Side Effects of Five Different Oil-based Adjuvants in Mice. Vet. Immunol. Immunopathol. 1998, 61, 291–304. (7) Murhekar, M. V.; Dutta, S.; Kapoor, A. N.; Bitragunta, S.; Dodum, R.; Ghosh, P.; Swamy, K. K.; Mukhopadhyay, K.; Ningombam, S.; Parmar, K.; Ravishankar, D.; Singh, B.; Singh, V.; Sisodiya, R.; Subramanian, R.; Takum, T. Frequent Exposure to Suboptimal Temperatures in Vaccine Cold-Chain System in India: Results of Temperature Monitoring in 10 States. Bull. World Health Organ. 2013, 91, 906–913. (8) Sloat B. R.; Sandoval M. A.; Cui, Z. Towards Preserving the Immunogenicity of Protein Antigens Carried by Nanoparticles while Avoiding the Cold Chain. Int. J. Pharm. 2010, 393, 197–202. (9) Singh, M.; O'Hagan, D. T. Advances in Vaccine Adjuvants. Nat. Biotech. 1999, 17, 1075–1081. (10) Reed, S. G.; Orr, M. T.; Fox, C. B. Key Roles of Adjuvants in Modern Vaccines. Nat. Med. 2013, 19, 1597–1608. (11) Coors, E. A.; Seybold, H.; Merk, H. F.; Mahler, V. Polysorbate 80 in Medical Products and Nonimmunologic Anaphylactoid Reactions. Ann Allergy Asthma Immunol. 2005, 95, 593–205. (12) Huang, C. Y.; Huang, M. H. Emulsifying Properties and Degradation Characteristics of Bioresorbable Polymeric Emulsifiers in Aqueous Solution and Oil-in-Water Emulsion. Polym. Degrad. Stabil. 2017, 139, 138–142. (13) Smidrkal, J.; Cervenkova, R.; Filip, V. Two-Stage Synthesis of Sorbitan Esters, and Physical Properties of the Products. Eur. J. Lipid Sci. Technol. 2004, 106, 851–855.


22

Page 23 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(14) MacDonald, R. C.; MacDonald, R. I.; Menco, B. P.; Takeshita, K.; Subbarao, N. K.; Hu, L. R. Small-Volume Extrusion Apparatus for Preparation of Large, Unilamellar Vesicles. Biochim. Biophys. Acta. 1991, 1061, 297–303. (15) Huang, C. H.; Huang, C. Y.; Cheng, C. P.; Dai, S. H.; Chen, H. W.; Leng, C. H.; Chong, P.; Liu, S. J.; Huang, M. H. Degradable Emulsion as Vaccine Adjuvant Reshapes Antigen-Specific Immunity and Thereby Ameliorates Vaccine Efficacy. Sci. Rep. 2016, 6, 36732. (16) Wu, H.; Zhu, H.; Zhuang, J.; Yang, S.; Liu, C.; Cao, Y. C. Water-Soluble Nanocrystals Through Dual-Interaction Ligands. Angew. Chem. Int. Ed. Engl. 2008, 47, 3730– 3734. (17) Yesilyurt, V.; Webber, M. J.; Appel, E. A.; Godwin, C.; Langer, R.; Anderson, D. G. Injectable Self-Healing Glucose-Responsive Hydrogels with pH-Regulated Mechanical Properties. Adv. Mater. 2016, 28, 86–91. (18) Reddy, S. T.; Swartz, M. A.; Hubbell, J. A. Targeting Dendritic Cells with Biomaterials: Developing the Next Generation of Vaccines. Trends Immunol. 2006, 27, 573–579. (19) Capek, I. Degradation of Kinetically-Stable O/W Emulsions. Adv. Colloid. Interface Sci. 2004, 107, 125–155. (20) Lin, S. I.; Huang, M. H.; Chang, Y. W.; Chen, I. H.; Roffler, S.; Chen, B. M.; Sher, Y. P.; Liu, S. J. Chimeric Peptide Containing both B and T Cells Epitope of Tumor-Associated Antigen L6 Enhances Anti-Tumor Effects in HLA-A2 Transgenic Mice. Cancer Lett. 2016, 377, 126–133. (21) Irvine, D. J.; Hanson, M. C.; Rakhra, K.; Tokatlian, T. Synthetic Nanoparticles for Vaccines and Immunotherapy. Chem. Rev. 2015, 115, 11109–11146.


23


Page 24 of 31

(22) Lai, C. H.; Tang, N.; Jan, J. T.; Huang, M. H.; Lu, C. Y.; Chiang, B. L.; Huang, L. M.; Wu, S. C. Use of Recombinant Flagellin in Oil-in-Water Emulsions Enhances HemagglutininSpecific Mucosal IgA Production and IL-17 Secreting T Cells against H5N1 Avian Influenza Virus Infection. Vaccine 2015, 33, 4321–4329. (23) Xia, Y.; Wu, J.; Wei, W.; Du, Y.; Wan, T.; Ma, X.; An, W.; Guo, A.; Miao, C.; Yue, H.; Li, S.; Cao, X.; Su, Z; Ma, G. Exploiting the Pliability and Lateral Mobility of Pickering Emulsion for Enhanced Vaccination. Nat. Mater. 2018, 17, 187–194. (24) Brito Baleeiro, R.; Schweinlin, M.; Rietscher, R.; Diedrich, A.; Czaplewska, J. A.; Metzger, M.; Michael Lehr, C.; Scherließ, R.; Hanefeld, A.; Gottschaldt, M.; Walden, P. Nanoparticle-Based Mucosal Vaccines Targeting Tumor-Associated Antigens to Human Dendritic Cells. J. Biomed. Nanotechnol. 2016, 12, 1527–1543.


24

Page 25 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Schematic illustration of a colloidal vesicle contoured by polymeric bioresorbable amphiphiles. Colloid consisting of two immiscible liquids, oil and water, was stabilized by amphiphiles consisting of hydrophilic groups and lipophilic groups. The hydrophilic group was made of PEG, sorbitan or ethoxylated sorbitan; on the other hand, the lipophilic group comprised either fatty acids or polyesters. The latter was derived from lactyl and caproyl monomotifs, which have been widely regarded degradable polyesters. These polymeric bioresorbable amphiphiles act as surface-active agents to stabilize the oil/water interfaces and give rise to oilshelled polysorbasomes (polymeric absorbable vesicles) during preparation and storage. The multiple phase character prolongs the retention of bioactive molecules. The degradability intrinsic to bioresorbable amphiphiles complies the vehicles' being destroyed and further being absorbed in vivo. These features provide new insights into the field of vaccine delivery based on bioresorbable amphiphilic engineering.


25


Page 26 of 31

Figure 2. Synthesis scheme for PEGylated polyester/sorbitan polyester copolymers. For sorbitan polyester synthesis, sorbitan was first obtained by phosphoric acid-catalysed anhydrization of sorbitol. Then, it was allowed to react with lactide (LA) and ε-caprolactone (ε-CL) in the presence of SnOct2, resulting in a sorbitan-PLACL copolymer composed of a hydrophilic sorbitan moiety and lipophilic PLACL segments. For PEGylated polyester synthesis, sorbitan was replaced with polyethylene glycol monomethyl ether (MePEG) to yield the block copolymer PEG-PLACL.


26

Page 27 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Characterization of a sorbitan-polyester copolymer (sorbitan-PLACL 1:4:4). (A) DOSY NMR spectra of sorbitan-PLACL in DMSO-d6. The proton NMR spectrum exhibits signals corresponding to the sorbitan moiety, PLA blocks, and PCL blocks. The three different blocks exhibited the same diffusion coefficient, suggesting efficient ligation of the sorbitan to the PLACL copolymers. (B) SEC chromatogram. Sorbitan-PLACL samples were dissolved in THF and measured by SEC. (C) Mass spectrometry measurements. An aliquot (1 µl) of sample was dried to form a matrix/analyte complex and analyzed by MALDI-TOF in reflection mode. Lowmolecular-weight components were measured on a plate containing porous silicone spots without matrix.


27


Page 28 of 31

Figure 4. Physicochemical characteristics of polysorbasomes. (A) Sustained-release depot for protein. Cumulative release of OVA from the designed PSS formulations at 37°C. Data are shown as mean ± s.d. from three samples. (B) Soft matter with tunable size. Homogeneous nanoparticles with an average diameter of 100 nm were observed after they were progressively passed through extrusion membranes. (C) Molecular weight changes of each component. In vitro degradation was performed in PBS at 37°C. Samples were freeze-dried, redissolved in THF, and applied to SEC. (D) Degradation-oriented disintegration. Visual aspects upon storage at 4°C, 25°C, or 37°C during the 26-week period. (B-D) Polysorbasome was stabilized by PEGPLACL/sorbitan-PLACL 1:4:4.


28

Page 29 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Depot effect of a polysorbasome (PSS). (A-B) PSS prolongs antigen retention and drives antigen-specific humoral immunity in vivo. (A) OVA* distribution in mice. IVIS fluorescence images of BALB/c mice (n = 3 per group) vaccinated with OVA* with or without PSS in both quadriceps at different time points. (B) Impact of PSS on driving OVA-specific IgG antibodies in mouse serum (n = 6 per group) treated either with free OVA protein or PSSformulated OVA (geometric mean ± s.d.; two-tailed Student's t-test of log10-transformed values, *p < 0.01). (C) Absorbance of PSS. Histological images (H&E staining) of the injection site at weeks 1, 3, and 7 following injection of PSS (original magnification, ×40 and ×400). Violet signals indicate the nuclei of infiltrated cells (yellow arrows). Data are representative of two independent experiments. PSS was stabilized by PEG-PLACL/sorbitan-PLACL 1:4:4.


29


Page 30 of 31

Figure 6. Modulation of cell-mediated immunity. C57BL/6 mice (n = 3 per group) were injected once subcutaneously with 10 µg/ml OVA alone or formulated with O/W or PSS. (A) APC phenotyping. Three days after vaccination, the draining LN cells were harvested from the inguinal and popliteal LNs, and the expression levels of MHC class II, CD40 and CD86 on the gated CD11c+ cells were determined by flow cytometry. (B,C) T-cell immunity. Seven days after vaccination, splenocyte suspensions were pooled and restimulated with 10 µg/ml OVA protein for 72 h. (B) Cytokine release profiles were measured in the supernatants collected from triplicate cultures, and subjected to ELISA paired antibodies. Data are shown as the cytokine concentrations in spleen cell restimulated with OVA antigen minus those with culture medium alone (mean ± s.d.). *p < 0.01. (C) The expression levels of transcriptional factors T-bet, RORγt, and β-actin (the reference standard) in OVA-restimulated splenocytes were recorded by RT-PCR. Data are representative of four independent experiments. PSS was stabilized by PEGPLACL/sorbitan-PLACL 1:4:4.


30

Page 31 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Graphical Abstract


31

Polysorbasome: a Colloidal Vesicle Contoured by Polymeric

Recommend Documents