Synthetic polymeric mixed micelles target to lymph node triggering

Subscriber access provided by UNIVERSITY OF ADELAIDE LIBRARIES

Article

Synthetic polymeric mixed micelles target to lymph node triggering enhanced cellular and humoral immune responses Chenxi Li, Xiaoxu Zhang, Qing Chen, Jiulong Zhang, Wenpan Li, Haiyang Hu, Xiuli Zhao, Mingxi Qiao, and Dawei Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b14004 • Publication Date (Web): 29 Dec 2017 Downloaded from http://pubs.acs.org on December 29, 2017

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 free 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 accessible to all readers and 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.

ACS Applied Materials & Interfaces 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 40 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

Synthetic polymeric mixed micelles target to lymph node triggering enhanced cellular and humoral immune responses

Chenxi Lia, Xiaoxu Zhanga, Qing Chena, Jiulong Zhanga, Wenpan Lia, Haiyang Hua, Xiuli Zhaoa, Mingxi Qiaoa*, Dawei Chena*

*Corresponding author: Email: [email protected]; [email protected] a

Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University,

Shenyang 110016, PR China

Key words Polymeric mixed micelles; Lymph node; Surface functionalization; Dendritic cell targeting; Endosomal rupture; Immune responses Abstract It has been widely accepted that lymph nodes (LNs) are critical targets of cancer vaccines since antigen presentation and initiation of T cell-mediated immune responses occur primarily at these locations. In this study, amphiphilic diblock copolymer PEOz-PLA combined with carboxylterminalted-Pluronic F127 were used to construct mixed micelles (carboxylated-NPs) for co-delivery

of

antigen

OVA

and

Toll-like

receptor-7

agonist

CL264

(carboxylated-NPs/OVA/CL264) to the LN resident DCs. The results showed that the small, sub-60nm size of the self-assembled mixed micelles enables them to rapidly penetrate into lymphatic vessels and reach DLNs after subcutaneous injection. Furthermore, the surface modification with carboxylic groups imparted the carboxylated-NPs with endocytic receptor targeting ability, allowing for DCs internalization of carboxylated-NPs/OVA/CL264 via scavenger receptor-mediate pathway. Because stimulation of CL264 in early endosomes will lead to a more effective immune response than that in late endo/lysosomes, the mass ratio of PEOz-PLA to carboxylated-Pluronic F127 in the mixed micelles was adjusted to release the encapsulated CL264 1

ACS Paragon Plus Environment

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 40

to the early endosome, resulting in increased the expression of co-stimulatory molecules and secretion of stimulated cytokines by DCs. Moreover, the incorporation of PEOz outside micellar shell effectively augmented MHC I antigen presentation through facilitating endosome escape and cytosolic release of antigens. This in turn evoked potent immune responses in vivo, including activation of antigen-specific T cell responses, production of antigen-specific IgG antibodies and the generation of cytotoxic T lymphocytes responses. Finally, immunization with the co-delivery system in E.G7-OVA tumor-bearing mice could not only significantly inhibit tumor growth, but also

markedly

prolong

the

survival

of

tumor-bearing

mice.

Taken

together,

carboxylated-NPs/OVA/CL264 has been demonstrated great potential for clinical applications as an effective anti-tumor vaccine for further immunotherapy. 1. Introduction Vaccine delivery systems targeting to lymph nodes (LN) has gained great significance due to the boosting of anti-tumor immune response1. Even though considerable efforts have been made to develop various nanoparticles to improve LN accumulation, only a few nanoparticle-based delivery systems could reach the lymphoid tissue. Moreover, the clinical efficacy has been rather disappointing because targeting LN by nanoparticles mostly boosted the IgG response rather than antigen-specific CD4+ and CD8+ T cell responses, which are considered to be crucial for effective cancer vaccine efficacy2-4. Therefore, it is highly demanded to develop new nanoparticle-based vaccine delivery systems that can maximize LN targeting and boost or induce the desire immune responses. Nanoparticle (NP) characteristics such as size, shape, and surface properties significantly affect biological processes in vivo5-7. NPs less than 5nm diffuse much faster into blood than those over 100nm, which showed poor lymphatic uptake and remain longer at the injection site. NPs with particle size ranging from 20 to 70 nm are optimal for rapid entry into lymphatic vessels and migration into lymph nodes. In addition, PEGylation of particles acts as an alternative approach to enhance lymphatic trafficking, as the PEG coating can reduce the non-specific interactions of particles with collagen fibers, glycosaminoglycans and proteins in the interstitium. NPs with hydrophilic surface diffused through the interstitial water channels more effectively than NPs with hydrophobic surface8-10. Previous studies indicated that the surface characteristics of nanoparticle delivery system had a profound effect on the driving differential T cell immunity11. For example, 2


Page 3 of 40 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


immunization with hyaluronic acid (HA)-decorated cationic lipid-poly(lactide-co-glycolide) acid (PLGA) hybrid nanoparticles (HA-DOTAP-PLGA NPs) was able to induce stronger CD4+ and CD8+ T cell responses than that with DOTAP-PLGA NPs because the degradation of HA in endosome could promote cytosolic release of antigens and subsequent antigens presentation on major histocompatibility complex (MHC) I pathway12. These findings highlighted the importance of optimizing the properties of nanoparticle-based vaccine delivery systems and this is considered to be a crucial part of strategy to enhance anti-tumor immune responses. The dendritic cells (DCs) in LNs exist in two functionally and phenotypically distinct stages as immature (imDCs) and mature DCs (mDCs)13-14. imDCs have better antigen uptake and processing ability, however mDCs dramatically downregulate their capability for non-specific uptake during maturation14. Therefore, utilizing appropriate endocytic receptors to facilitate vaccine internalization in DCs is essential. A multitude of surface receptors displayed on DCs have been investigated for active targeting, including mannose receptor (MR), DC-SIGN, scavenger receptor (SR), DEC-205, and Toll-like receptors15-18. Some polymers that inherently possess endocytic receptors targeting property offers an alternative approach to enhance vaccine internalization such as Pluronics. For example, water-in-oil or oil-in-water emulsions formulated with selected Pluronic block copolymers have been used as a carrier to facilitate antigen molecules uptake by antigen present cells (APCs)19. Furthermore, Pluronics can directly incorporate into membranes followed by subsequent translocation into the cells, which can significantly enhance the efficiency of vaccine platform penetrating into cells20-21. In

this

study,

the

amphiphilic

diblock

copolymer

of

poly(2-ethyl-2-oxazoline)-poly(D,L-lactide) (PEOz-PLA) and carboxylterminated-Pluronic F127 were used to construct mixed micelles (COOH-Pluronic F127/PEOz-PLA, denoted as carboxylated-NPs)

targeting

LN

resident

DCs

for

vaccine

delivery.

The

carboxylterminated-Pluronic is superior in cellular uptake because it could share scavenger receptor-mediated delivery as compared to other Pluronic derivatives. Scavenger receptors are able to recognize a variety of negatively charged substances or surfaces such as aged erythrocytes or apoptotic cells on macrophages and DCs22-24. PEOz is the hydrophilic block of the amphiphilic copolymer, which forms hydrophilic shell of the micelles. PEOz has similar hydrophilicity property with PEG, however, the tertiary amine on PEOz backbone can be protonated under the 3



acidic conditions which makes them possess endosomal escape capacity25-26. Herein, the design and preparation of these mixed micelles were described, and the effect of surface properties of micelles on the targeting of nanoparticles to draining lymph node (DLNs) and vaccine delivery efficiency as well as antigen-specific immune responses were investigated thoroughly. The mixed micelles of OH-Pluronic F127/PEOz-PLA (denoted as hydroxylated-NPs) and COOH-Pluronic F127/OH-Pluronic F127 (denoted as Pluronic-NPs) were constructed as control. In addition, we loaded the micelles with model antigen ovalbumin (OVA) and Toll-like receptor 7 (TLR-7) agonist CL264 that has been shown to stimulate mammalian immune cells with substantially higher potency than Imiquimod27. After subcutaneous injection of this mixed micelles (denoted as carboxylated-NPs/OVA/CL264), as outlined in the schematic shown in Fig. 1B, it is expected that carboxylated-NPs/OVA/CL264 could efficiently flow to the DLNs and target not only imDCs but also mDCs. The endo-lysosomal escape property of the PEOz is also expected to promote antigen cross-presentation via MHC I pathway, eliciting the potent T cell immunity against tumor cells. Therefore, it is believed that this versatile mixed micellar system can be utilized as a promising platform for anti-tumor immunotherapy.

4


Page 4 of 40

Page 5 of 40 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


Fig.1. Schematic illustration of synthetic mixed micelles targeting to LN resident DCs for anti-tumor immunotherapy. A) Schematic diagram shows the formulation of OVA-conjugated CL264-loaded

mixed

micelles.

carboxylated-NPs/OVA/CL264

in

B)

The

vivo.

hypothesis The

of

optimally

the

immune

engineered

function size

of

enables

carboxylated-NPs/OVA/CL264 efficiently accumulate in DLNs after subcutaneous injection and promote DCs including mDCs uptake via scavenger receptor-mediate endocytosis. The endo-lysosomal escape property effectively augments MHC I antigen presentation through facilitating cytosolic antigen release and hence induces potent cellular immunity against tumor. 2. Materials and methods 2.1. Materials Pluronic F127, 2-Ethyl-2-oxazoline (EOz),

D,L-lactide,

stannous octoate, Ovalbumin(OVA) ,

OVA257 264, and OVA323 339 were purchased from Sigma-Aldrich (MO, USA). p-toluene sulfonyl −

−

chloride, mercaptopropionic acid methyl ester, cysteamine·HCl, aldrithiol, N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC·HCl) were obtained from J&K scientific Ltd. (Beijing, China). Hoechst 33258 and LysoTracker Red were purchased from Invitrogen (USA). The mouse cytokine ELISA kits, fluorochrome-labeled CD11c, CD4, CD40, CD80, CD86, CD8 and CD3 were purchased from eBioscience (San Jose, CA,USA). BD Cytofix/Cytoperm Plus (with GolgiPlug) were purchased from BD Biosciences (San Jose, CA, USA). Rhodamine-labeled CL264 was purchased from InvivoGen (San Jose, CA, USA). All other chemical and buffer solution were analytical grade. 5



Page 6 of 40

Tested formulations: carboxylated-NPs: mixed micelles composed of COOH-Pluronic F127 and PEOz-PLA; hydroxylated-NPs: mixed micelles composed of OH-Pluronic F127 and PEOz-PLA; Pluronic-NPs: mixed micelles composed of OH-Pluronic F127 and COOH-Pluronic F127; carboxylated-NPs/OVA+CL264 polyplex: the physical mixture of free CL264 and the OVA loaded-carboxylated-NPs;

carboxylated-NPs/OVA:

OVA

loaded

carboxylated-NPs;

hydroxylated-NPs/OVA: OVA loaded hydroxylated-NPs; Pluronic-NPs/OVA: OVA loaded Pluronic-NPs; carboxylated-NPs/OVA/CL264: OVA and CL264 co-loaded carboxylated-NPs; hydroxylated-NPs/OVA/CL264:

OVA

and

CL264

co-loaded

hydroxylated-NPs;

Pluronic-NPs/OVA/CL264: OVA and CL264 co-loaded Pluronic-NPs. 2.2

Synthesis

and

characterization

of

PEOz-PLA,

carboxylated-Pluronic

F127

and

OVA-ss-Pluronic F127 copolymer. The copolymers used in this work were home-made. The details of synthesis and characterization of copolymers were shown in the Supplementary data. 2.3 Preparation and characterizations of OVA-conjugated CL264-loaded mixed micelles The carboxylated-NPs/OVA/CL264 was prepared by dialysis method. 1.5 mg CL264 was dissolved in 3 mL DMSO followed by the addition of 25mg copolymer mixtures consisting of PEOz-PLA, COOH-Pluronic F127 and OVA-ss-Pluronic F127 under agitation. The mixture stirred at room temperature overnight and then 10 mL PBS was added dropwise with a syringe. After DMSO was removed by dialysis against 3×1 L of Milli-Q water for 8 h, the solution was collected and freeze-dried, and mixed micelles were obtained. The hydroxylated-NPs/OVA/CL264 was prepared as described above without adding COOH-Pluronic F127. For the preparation of Pluronic-NPs/OVA/CL264 mixed micelles, identical operation was conducted except that the equivalent weight ratio of PEOz-PLA was replaced by OH-Pluronic F127 and the FITC-labeled mixed micelles was prepared as the same to described above except that the OVA-ss-Pluronic F127 was replaced by FITC-OVA-ss-Pluronic F127. To determine the amount of CL264 encapsulated in the mixed micelles, 2 mg of CL264-loaded mixed micelles were dissolved in 2 mL of DMSO under vigorous vortexing and the CL264 content was measured using high performance liquid column (HPLC). The particle size, size distribution and the zeta-potential of mixed micelles were all analyzed by a Zetasizer (Nano ZS,Malvern Co. Ltd, UK). Morphology of mixed micelles was observed by a 6


Page 7 of 40 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


transmission electron microscopy (TEM) (TecnaiG220, FEI, USA). The stability of carboxylated-NPs/OVA/CL264 in the presence of RPMI 1640 with FBS was evaluated by nonreducing SDS-PAGE. Carboxylated-NPs/OVA/CL264 was incubated in RPMI 1640 containing 10% FBS at 37℃ for the different time points, then the stability of mixed micelles was tested via nonreducing SDS-PAGE and the corresponding diameter was evaluated by dynamic light scattering (DLS). The CMC value of mixed micelles were determined using fluorescence spectrophotometry with pyrene as a probe. Briefly, pyrene solution (6.0 × 10-5 M) was dropped into brown volumetric flask and the solution was removed under a nitrogen flow. Then copolymer mixed micellar solutions with concentrations ranged from 0.5 to 100 mg/mL-1 was added into each brown volumetric flask to obtain the final pyrene concentration of 6.0 × 10-7 M. These solutions allowed to stock in the dark overnight and the fluorescence excitation spectra were measured using fluorescence spectrophotometer (LS55, PerkinElmer, USA). Excitation spectrum was monitored from 300 to 360 nm and the emission wavelength was set at 390nm. The fluorescence intensity ratio of I336/I334 was used to quantify the CMC value of mixed micelles. The DSC thermograms of the lyophilized powders of PEOz-PLA, COOH-Pluronic F127, the physical mixture of two copolymers and the mixed micelles were measured using METTLER TOLEDO DSC1 and the temperature was scanned range from 25℃ to 200℃ at the heating rate of 10℃/min. 2.4 pH-dependent drug release from the mixed micelles The CL264 released from mixed micelles was conducted using the dialysis method. Briefly, 2 mL of CL264-loaded mixed micelles solution was sealed in a dialysis bag (MWCO 14,000) and incubated in a 50 mL of phosphate buffer solution (PBS) with different pH values ( pH 7.4 or pH 5.5) at 37℃ under sink conditions. 1 mL sample was taken at different time point and replaced with the equal volume of PBS. The CL264 concentration in PBS solution was measured by HPLC. 2.5 Generation of murine bone-marrow derived DC (BMDC) Bone marrow cells were collected from femurs and tibiae of C57BL/6 mice. After washing in RPMI 1640 media, red blood cells were lysed with NH4Cl buffer and the collected bone marrow cells were seeded in a 6-well plates with 4mL of DC complete medium (RPMI-1640 with penicillin−streptomycin, 10% heat-inactivated FBS supplemented with 10 ng/mL of GM-CSF). On 7



Page 8 of 40

day 3, culture medium was removed and replaced with the fresh one. On day 6, the nonadherent and loosely adherent cells were harvested. The expression level of CD11c measured by flow cytometry showed that the purity of the DC population was between 80 and 85%. 2.6 In vitro cellular uptake To determine the intracellular delivery of mixed micelles, BMDCs were incubated with carboxylated-NPs/OVA/CL264, hydroxylated-NPs/OVA/CL264, Pluronic-NPs/OVA/CL264 and physical mixture of free OVA +CL264 (four formulations of nanovaccine all containing 50µg/mL OVA and 4µg/mL CL264) in a 6-well plate at 37℃for 6 h. After washing with PBS, the cells were collected for analysis using flow cytometry. To investigate the mechanism of uptake, cells were pre-incubated

in

the

presence

of

dextran

sulfate

followed

by

the

addition

of

carboxylated-NPs/OVA/CL264 for 1 h. 2.7 Cytokine and maturation assay BMDCs

were

seeded

hydroxylated-NPs/OVA/CL264,

in

a

6-well

plate,

then

carboxylated-NPs/OVA/CL264,

Pluronic-NPs/OVA/CL264,

carboxylated-NPs/OVA+CL264

polyplex and the mixture of free OVA+CL264 (different formulations of nanovaccines all containing 50µg/mL OVA and 4µg/mL CL264) were added to the wells in a 2mL total volume. The BMDCs were stained with anti-mouse CD40 and anti-mouse CD86 after 24h treatment. Control was not treated with antibody and the expression of CD40 and CD86 on BMDCs was measured by flow cytometry (BD FACSCalibur System). Furthermore, the culture supernatants were also harvested and the amount of IL-2p70 and TNF-ɑ was quantified using cytokine-specific ELISA according to the manufacturer’s instructions. Cytokine concentrations were quantified under a BioRed microplate reader (MK3, Thermo, USA) with the detection wavelength of 450 nm. 2.8 In vitro antigen cross-presentation assay. In vitro cross-presentation assay was evaluated using CD8-OVA 1.3 T cells, which could produce IL-2 upon recognition of MHC class I/OVA-derived peptide complexes. BMDCs were cultured in a 6-well plate with different formulations of nanovaccine at 50µg/mL OVA concentration at 37℃ for 4h. After washing three times with PBS, BMDCs were co-cultured with CD8-OVA1.3 cells for 24h. The amount of IL-12 released into medium was determined by IL-2 ELISA Kit according to the manufacture's instruction. 8


Page 9 of 40 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


To detect the intracellular localization of above nanovaccines, BMDCs were stained with LysoTracker Red for 1 h to visualize the endo-lysosomes. The cells were fixed with 4% paraformaldehyde for 15min after washing with cold PBS and stained subsequently with Hoechst 33258 to visualize the nucleus. Finally, the coverslips were mounted on a glass slides using Confocal laser scanning microscopy (CLSM) to determinate the subcellular localization. 2.9 In vivo transport of nanovaccines into DLNs C57BL/6 mice were subcutaneous injected with FITC-OVA/rhodamine B-CL264 loaded hydroxylated-NPs, Pluronic-NPs, carboxylated-NPs or the physical mixture of OVA+CL264. 3h after administration, inguinal LNs were carefully isolated and the FITC+/rhodamine B+ double positive cells were analyzed by flow cytometry. To determine the above nanovaccines in DCs, immune cells from DLNs were stained with APC-CD11c+ antibody for 30 min at 4℃, and then the FITC+/APC+ double positive cells were assessed by flow cytometry. 2.10 Immunization of mice C57BL/6 mice aged 6–8 weeks were randomly divided into 5 groups, which were immunized on day 0, 7 and 14 with one of the following formulations (all containing 50µg OVA and 50µg CL264) (n= 5 per group): carboxylated-NPs/OVA/CL264, hydroxylated-NPs/OVA/CL264, Pluronic-NPs/OVA/CL264, physical mixture of free OVA+CL264 or PBS. Subcutaneous immunizations were delivered in 200uL every time. 2.11 Preparation of splenocyte culture. Mice were sacrificed one week after last immunization (on day21) and spleens from each experiment group were collected and mechanically digested into single-cell suspensions in complete RPMI 1640 medium. The obtained cell suspension was then filtered through a 100µm cell strainer to remove the residual tissue fragments. Finally, cells were washed twice and resuspended in 1640 medium complete culture medium. 2.12 Assessment of adaptive immune response Splenocytes (2 × 106 cells) from each experimental group were seeded in a 6-well plate and cultured in the presence of OVA257−264 (100 µg/mL) or OVA323−339 (100 µg/mL) at 37℃. The cells were

then

washed

with

PBS

and

stained

with

APC-anti-CD3e/PE-anti-CD8a

or

APC-anti-CD3e/PE-anti-CD4 for 30 min at 4℃. The cells were washed with PBS twice and permeabilized for 20 min at 4℃ using the Cytofix/Cytoperm™ Plus Fixation/Permeabilization kit 9



according to the manufacturer's instructions. The percentage of IFN-γ+ CD4+ T cells and IFN-γ+ CD8+ T cells were measured by flow cytometry. 2.13 Antibody production One week after the last immunization, mice were sacrificed and approximately 50µL of blood was collected from eyeball for testing antigen-specific total lgG, IgG1 and IgG2a. Microplates were coated with (0.5 µg/100 µL) OVA at 4℃ overnight. After washing three times, the plates were block with BSA solution at 37℃for 2 h. Then sera were serially diluted and incubated in the plates at room temperature for 2h. The OD450 was measured by a microplate reader. 2.14 CTL assay The CTL assay was performed using a calcein-AM release assay. Briefly, the isolated splenocytes from each experimental groups were re-stimulated with 100µg/mL OVA257−264 for 3 days. E.G7-OVA cells acted as target cells were labeled with 5 uM calcein-AM at 37°C for 30 min. Then the effector cells incubated with target cells at varied ratios of 10:1,20:1,40:1 and 80:1 at 37℃ for 4 h and the specific lysis was analyzed by fluorescence spectrophotometer (excitation wavelength at 485 ± 9nm and the emission wavelength at 530 ± 9nm) using equation. Specific lysis (%) = test release-spontaneous release / maximum release-spontaneous release

×100. 2.15 In vivo tumor challenge 2×105 E.G7-OVA cells were inoculated subcutaneously into the right back of C57BL/6 mice on day 0 and then immunized with PBS or 50ug OVA and 50ug CL264 in various formulations of nanovaccine on day 5, 11 and 17. Mice body weight and tumor size were monitored every 3 days and the tumor size was measured using following formula: tumor volume (mm3) = length × (width2) × 0.5. 2.16 Statistical analysis All data in this paper are shown as mean ± SD (n = 3-5). The significance of the data was evaluated using Student’s t test; a p-value < 0.05 was considered significant. 3. Results 3.1 Characterization of synthetic polymeric mixed micelles Carboxylated-NPs/OVA/CL264 that can co-deliver antigens and immunostimulatory adjuvants to the LNs were developed. The optimum mass ration of PEOz-PLA to COOH-Pluronic 10


Page 10 of 40

Page 11 of 40 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


F127 was determined depending on the intracellular pH-dependent CL264 release. It was reported that the internalization of TLR7 ligands in early endosomes will lead to a more effective immune response than that in late endo/lysosomes28. Therefore, to exert its immunomodulatory effects, the encapsulated CL264 must be released from the NPs when it is internalized into the early endosomes29. The pH-sensitive delivery systems are required to have a proper triggering pH value, at which a remarkable accelerated drug release occurs and this pH value is pHt30. The pHt of the mixed micelles composed of different mass ratios of PEOz-PLA/COOH-Pluronic F127 was investigated and the results showed that when PEOz-PLA to COOH-Pluronic F127 at the mass ratio of 65/35, the mixed micelles showed a desirable pHt (~5.5), which was specifically responsive to the early endosomal pH. The typical pH-dependent CL264 release profile from the mixed micelles at the weight ratio of 65/35 was shown in Fig.2B. The preparation of CL264-loaded OVA-conjugated mixed micelles (carboxylated-NPs/OVA/CL264) was performed by

dialysis

method.

As

the

controls,

hydroxylated-NPs/OVA/CL264

and

Pluronic-NPs/OVA/CL264 were prepared as the identical operation except that the equivalent weight ratio of COOH-Pluronic F127 or PEOz-PLA was replaced by OH-Pluronic F127. The particle size and size distribution were characterized by dynamic light scattering (DLS) and the mean diameters of three mixed micelles were all sub-60nm, which exhibited the excellent size for transit through lymphatic vessels to the lymph nodes. The transmission electron microscopy (TEM) images showed that the micelles have a spherical and homogeneous morphology with narrow size distribution, and the sizes of the mixed micelles from TEM image were consistent well with those obtained by DLS. The ability of mixed micelles to carry OVA was evaluated by SDS-PAGE and the conjugation efficiency was approximately＞90% according to the BCA protein assay, giving 0.069 mg of conjugated OVA per mg of mixed micelles. Since the particle charge plays an important role in the scavenger receptor-mediated endocytosis, the zeta-potential of carboxylated-NPs/OVA/CL264 was hence measured and the results showed that it was a negative charge (-20.7mV) due to the ionization of carboxylic group in the micellar shell under physiological condition and the number of negative charges was enough to be recognized by scavenger receptors31. The physical characterizations of the mixed micelles were summarized in Table 1. The CMC value is an important parameter of the micelle, which involves the self-assembly 11



Page 12 of 40

ability of the amphiphilic copolymer and influence the structural stability of micelles in vitro and in vivo32. The CMC value of three mixed micelles were measured by fluorescence spectrophotometry with pyrene as a hydrophobic fluorescence probe. The fluorescence intensity ratio of I336/I334 remained at a low value at the low copolymer concentration and when the concentration reached a certain value, pyrene was solubilized into the hydrophobic core of micelles, resulting in a remarkable increase in the intensity ratio of fluorescence intensity ratio. As shown in Fig.S10, the CMCs of carboxylated-NPs, hydroxylated-NPs and Pluronic-NPs were 2.69, 6.6 and 14.1µg/mL, respectively. These relatively low CMC value could ensure the stability of mixed micelles in the extracellular matrix after subcutaneous administration. For induction of an effective immune response, the nanoparticle-based delivery system must be able to make sure that the antigen arrives at the site of action without being degraded or released prematurely33. To detect whether OVA was able to bound to the mixed micelles until they arrived at the LNs, the stability of the mixed micelles in the presence of complete medium with FBS for different periods of time was tested via nonreducing SDS-PAGE. As we expected, antigen OVA was still bound to the mixed micelles even under incubation in 10% FBS for 72h, implying carboxylated-NPs are able to maintain OVA conjugated onto themselves for a long time. Furthermore, the particle size of these three different micelles did not significantly unchanged during 72h incubation in FBS (Fig.2C), which further confirmed the great stability of mixed micellar nanovaccines. This remarkable stability is important for applications in vivo, which could ensure nanoparticles successfully traffic through the extracellular matrix of the lymphatic system after subcutaneous injection and deliver antigen to the immune cells in LNs. Table 1 Characterization of OVA-conjugated CL264-loaded mixed micelles. Formulation

carboxylated-NPs/OVA /CL264 hydroxylated-NPs/OVA /CL264 Pluronic-NPs/OVA /CL264 a

Ζ-potential

CL264 content

OVA content

Size (nm)a

PDI

54.2 ± 2.6

0.33 ± 0.01

-20.7 ± 1.5

66.7 ± 8.5

69.7 ± 10.3

50.4 ± 2.1

0.31 ± 0.02

-4.1 ± 0.3

63.2 ± 7.4

72.9 ± 12.4

45.2 ± 1.9

0.33 ± 0.05

-18.9 ± 0.7

65.3 ± 7.9

74.6 ± 9.7

(mv)

Data are presented as mean diameter ± SD, n=3 12


(µg/mg)

b

(µg/mg) b

Page 13 of 40 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


b

Loading amount per 1 mg of mixed micelles.

Fig.2. A) The TEM images of mixed micelles. Scale bar represents 100 nm. B) In vitro CL264 release from carboxylated-NPs/OVA/CL264 in different pH at 37 ℃ . C) Stability of mixed micelles in the presence of FBS at 37℃. (Data are presented as mean ± SD n = 3) 3.2 Co-micellization of PEOz-PLA and COOH-Pluronic F127 To prepare mixed micelles, one issue must be demonstrated that forming the micelles composed of the co-micellization of two mono-copolymer rather than the physical mixture of them30. Therefore, the mono-copolymer PEOz-PLA, mono-copolymer COOH-Pluronic F127, the physical mixture of two mono-copolymer and the mixed micelles were measured by DSC analysis. As shown in Fig.S11, mono-COOH-Pluronic F127 and mono-PEOz-PLA showed endothermic peak at around 51.51°C and 74.43°C, respectively. As expected, physical mixture of two mono-copolymers exhibited the exactly same endothermic peaks in the thermogram (COOH-Pluronic F127: 52.49°C and PEOz-PLA: 76.30°C), indicating the phase separation between the two kinds of micelles. However, the corresponding endothermic peaks in the 13



Page 14 of 40

thermogram of the mixed micelles shifted to 47.88°C and 67.98°C, respectively, which could be attributed to the strong interactions between PEOz-PLA and COOH-Pluronic F127 due to the co-micellization of PEOz-PLA and COOH-Pluronic F127. This result suggested that the obtained micelles was co-micellization micelles rather than the mixture of two mono-copolymer micelles. In addition, the hydrodynamic diameter (dh) and polydispersity index (PDI) of the mixed micelles at different weight ratios of PEOz-PLA to COOH-Pluronic F127 were also investigated. As shown in Fig.S12, the average particle size of the mixed micelles was around ~50nm when the weight ratio of PEOz-PLA/COOH-Pluronic F127 was 65/35, which fell into the window of PEOz-PLA mono-micelle (~60nm) and COOH-Pluronic F127 mono-micelle (~40 nm). The median particle size and the relatively narrow size distribution polydispersity of the mixed micelles further indicate the co-micellization of the two copolymers30. 3.3 Carboxylated-NPs enhance cellular uptake of OVA and CL264 The uptake of vaccine components by immune cells is the initial step for antigen-processing and immune activation. To determine whether carboxylated-NPs could act as an effective DC-targeting vaccine that can facilitate OVA and TLR7 agonist CL264 uptake, we incubated BMDCs

with


hydroxylated-NPs/OVA/CL264,

Pluronic-NPs/OVA/CL264 and physical mixture of free OVA and CL264 (hereafter referred to as OVA+CL264). All experimental nanoparticles were formulated with rhodamine-labeled CL264 and fluorescein isothiocyanate (FITC)-labeled OVA and the internalization by BMDCs were analyzed by fluorescence microscopy and flow cytometry. After 3h incubation, as shown in Fig.3A, the internalization of physical mixture of OVA+CL264 in BMDCs were detectable but low, suggesting free OVA and CL264 were unable to penetrate into cells efficiently. In contrast, co-delivery of OVA and CL264 via the form of nanoparticle can greatly enhance the internalization of OVA and CL264 in BMDCs. In addition, flow cytometry analyses showed that the enhancement observed in carboxylated-NPs/OVA/CL264 incubated BMDCs was much greater than

that

in

the

BMDCs


incubated had

with a

hydroxylated-NPs/OVA/CL264, higher

affinity

to

suggesting

BMDCs

than

hydroxylated-NPs/OVA/CL264. Since the scavenger receptor is well known for its involvement in the uptake of a variety of negatively

charged

particles,

we

speculated

that

the

14


enhanced

internalization

of

Page 15 of 40 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


carboxylated-NPs/OVA/CL264 in BMDCs may be ascribed to the scavenger receptor-mediated pathway. To confirm this, a competitive inhibition experiment using dextran sulfate22 which is inhibitors for scavenger receptors was conducted. The results showed that the pre-treatment with dextran sulfate partially inhibited the uptake of carboxylated-NP/OVA/CL264 by BMDCs, implying the scavenger receptor was indeed involved in the BMDCs uptake. Due to the similar composition of COOH-Pluronic F127 in the mixed micelles, Pluronic-NPs/OVA/CL264 showed almost the same cellular uptake behavior with carboxylated-NPs/OVA/CL264 in BMDCs. These data

suggested

that

the

high

performance

of


or

Pluronic-NPs/OVA/CL264 in cellular uptake benefits from the scavenger receptor-mediated endocytosis and the design of carboxylic groups located on the micelles shell is a key component to provide the mixed micellar nanoparticles with targeting ability.

Fig.3. Carboxylated-NPs/OVA/CL264 enhance OVA and CL264 uptake by BMDCs. BMDCs were incubated with various formulations of nanovaccine at a concentration of 50µg/mL OVA and 4µg/mL CL264 for 3h. The representative fluorescence microscopy images were shown after cellular uptake (A) and the cellular uptake efficiency was quantified by flow cytometry (B). (OVA labeled with FITC for green fluorescence and CL264 labeled with rhodamine B for red fluorescence) (MFI=mean fluorescence intensity). (Data are presented as mean ± SD n = 3 *P < 0.05


hydroxylated-NPs/OVA/CL264

and

Pluronic-NPs/OVA/CL264 vs OVA+CL264, #P < 0.05 carboxylated-NPs/OVA/CL264 and Pluronic-NPs/OVA/CL264 vs hydroxylated-NPs/OVA/CL264). (Scale bars represent 10 µm). 15



Page 16 of 40

3.4 Carboxylated-NPs/OVA/CL264 enhance BMDCs maturation and activation For T cell activation, DCs must express maturation markers such as adhesion-related (CD40, CD54) and costimulatory (CD80, CD86) molecules on their surface34. The expression of these maturation markers on DCs is enhanced when DCs are activated. Therefore, we next assessed whether carboxylated-NPs/OVA/CL264 had capabilities to effectively activate BMDCs and the expression of CD40 molecules and CD86 molecules were analyzed by flow cytometry with CD40-specific or CD86-specific antibodies. As shown in Fig.4A, carboxylated-NPs significantly increased expression levels of both CD40 and CD86 relative to the groups that were treated with physical mixture of OVA+CL264. Moreover, carboxylated-NPs/OVA+CL264 polyplex did not show similar enhancement in maturation markers expression when

compared with

carboxylated-NPs/OVA/CL264, suggesting that free TLR7 agonist had modest effect on BMDC maturation. It is well-known that TLR7 is expressed inside the endo-lysosomal compartments of DCs29, therefore, effective delivery of agonist into DCs is indispensable for activating TLR7 signaling and subsequent immune response35. Free agonist molecules hardly localize to TLRs, thus, carboxylated-NPs could act as an effective delivery carrier that could enter DCs via endocytosis and be internalized into the endo-lysosome. Furthermore, owing to the pH-sensitve properties of carboxylated-NPs, the acidification of the endo-lysosome caused dissociation of the micelles and then triggered agonist rapid release from the carboxylated-NPs, allowing it to engage its molecular target, TLR7. These results indicated that the improved immunostimulatory effect on BMDC maturation resulted from the enhanced delivery-capacity of carboxylated-NPs and CL264 combination in a cooperative way. The BMDC maturation was further confirmed through the cytokines production by BMDCs. IL-12p70 is known as an important pro-inflammatory cytokine for activating CD8+ T cells and TNF-ɑ is considered as a stimulating factor for initiating effective cellular immune responses12, 35-36

. As shown in Fig.4C-D, carboxylated-NPs/OVA/CL264 induced greater production of TNF-ɑ

and IL-12p70 than other groups and secretion of these Th1-polarizing cytokines were time-dependent. Such a sustained enhancement of cytokine production could be observed during the first few hours and reach a plateau at 12 to 24 hours, which can amplify the cellular immune response by efficiently activating the CD8+ T cells and potentiate cytotoxic killing of CD8+ T cells37. However, this phenomenon did not observe from Pluronic-NPs/OVA/CL264 treated 16


Page 17 of 40 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


BMDCs.

This

difference

in

cytokines

production

could

be

attributed

to

the

carboxylated-NPs/OVA/CL264 with endosomal pH triggered release property. In summary, it is confirmed that carboxylated-NPs/OVA/CL264 were effective at promoting BMDC activation and maturation.

17



Fig.4. Carboxylated-NPs/OVA/CL264 promote BMDC maturation and MHC-I antigen presentation. A-B) Expression of CD40 and CD86 molecules was measured by flow cytometry after 24h BMDCs incubation with various formulations of nanovaccine at a concentration of 50µg/mL OVA and 4µg/mL CL264. Representative flow cytometry histograms were shown and the median fluorescent intensity (MFI) for each treatment group was summarized in a histogram with a bar. The concentrations of IL-12p70 and TNF-ɑ cytokines in the culture medium were measured by ELISA kits (C-D). E) BMDCs were incubated with nanovaccines at 50µg/mL OVA concentration for 4h and subsequently co-cultured with CD8-OVA1.3 cells for 24h. The IL-2 released from CD8-OVA1.3 cells was measured by ELISA kits. F) CLSM images of BMDCs after 3 h incubation with FITC-labeled OVA-loaded nanovaccines. Nuclei were stained with Hoechst 33258 (blue) and endo-lysosomes were stained with LysoTracker Red. (Data are presented as mean ± SD n = 3 *P < 0.05 carboxylated-NPs/OVA/CL264, hydroxylated-NPs/OVA/CL264 and Pluronic-NPs/OVA/CL264 vs OVA+CL264 and carboxylated-NPs/OVA+CL264 polyplex, #P < 0.05 carboxylated-NPs/OVA and hydroxylated-NPs/OVA vs Pluronic-NPs/OVA). (Scale bars 18


Page 18 of 40

Page 19 of 40 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


represent 10 µm). 3.5 PEOz coating enhances antigen MHC I cross-presentation An effective vaccine designed to evoke anti-tumor immune responses requires the induction of antigen-specific cytotoxic (CD8+) T lymphocytes as well as T helper (CD4+) cells3-4. A prerequisite for activation CD8+ or CD4+ T cell is the presentation of antigen peptides through MHC class II or class I molecules by antigen-presenting cells (APCs). Traditional subunit vaccines endocytosed by APCs are usually processed in the endo/lysosomal compartments that generally preferred the MHC class II rather than MHC I antigen presentation38. To measure whether carboxylated-NPs could promote MHC class I antigen presentation, a coculture assay was conducted. BMDCs were incubated with various formulations of nanovaccine and co-cultured subsequently with CD8-OVA1.3 which produces IL-2 upon the recognition of MHC class I/OVA derived peptide complexes. As depicted in Fig.4E, a higher level of IL-2 production was observed from CD8-OVA1.3 cells when co-cultured with OVA-nanoparticle-conjugates than those co-cultured with free OVA-treated BMDCs, indicating that the reduction-sensitive conjugation of antigen onto particles could effectively promote the efficiency of antigen cross-presentation39. Furthermore, compared with Pluronic-NPs/OVA, carboxylated-NPs/OVA treated BMDCs induced more IL-2 production from co-cultured CD8-OVA1.3 cells, suggesting PEOz-coating plays an important role in promoting antigen presentation via MHC I pathway. This could be ascribed to the endosomolytic activity of PEOz. Since the amide groups on PEOz were ionized under acidic endo/lysosomes environment, leading to the increased electrostatic repulsion between PEOz blocks which may induce dissociation of the micelle structure followed by lysosomal rupture25-26. Thereby antigen molecules could escape from endo/lysosomal and degrade in cytosolic proteasomes for MHC I antigen presentation. We next used confocal imaging to further investigate the subcellular localization of mixed micelles in BMDCs by staining the endo-lysosomes. After Pluronic-NPs/OVA was incubated with BMDCs for 3h, as shown in Fig.4F, most of the FITC-OVA was colocalized with red fluorescence stained endo-lysosomes, which showed a yellow fluorescence in merged images, indicating that these mixed micelles mainly resided in end/lysosomes. While in the carboxylated-NPs/FITC-OVA treated cells, many FITC-OVA molecules were located in cytosol, showing the separation of red and green fluorescence and the FITC-OVA green fluorescence became stronger (white arrow). 19



This suggested that antigen molecules delivered by carboxylated-NPs were able to escape from lysosome into cytoplasm for cross-presentation. Here emerges a problem that even though a pH-responsive micellar system with strong endosomolytic activity was employed, part of the antigen molecules still failed to escape from endosome (yellow arrow). A similar phenomenon has been observed from antigen and oligonucleotides dual-delivery pH-sensitive nanoparticle (OVA-pol/CpG) 40. The reason described in previous studies have shown that the antigen delivered to the cytosol could be trafficked subsequently back to the endo/lysosomal compartments through autophagy40. Considerable of evidence have shown that the autophagy promotes cytosolic antigen presentation via MHC class II pathway. Therefore, it is believed that a part of cytosolic antigen experienced engulfment in the autophagosomes, which is then processed for MHC-II presentation Hence our mixed micelles carboxylated-NPs delivered antigen could presentation via both the MHC-I and MHC-II pathways. 3.6 Carboxylated-NPs efficiently target to the draining lymph nodes. Because co-delivery of antigen and immunostimulatory adjuvant to LNs is an effective strategy to enhance antigen-specific immune response8-10. Our mixed micelles had an optimal size for trafficking to LNs, therefore we next to evaluate whether carboxylated-NPs could be used to target LNs. FITC-OVA and rhodamine-CL264 co-loaded carboxylated-NPs/OVA/CL264, hydroxylated-NPs/OVA/CL264, Pluronic-NPs/OVA/CL264 and the physical mixture of free OVA+CL264 were injected into the tail base of C57BL/6 mice. At 3h post-injection, their inguinal LNs were collected and the isolated cells were analyzed by flow cytometry. As shown in Fig.5, mice injected with OVA and CL264 in nanoparticle forms showed significantly higher proportions of FITC+/ rhodamine B+ double-positive cells than those in a physical mixture of free OVA and CL264 injected mice, indicating the mixed micelles can efficiently deliver their payloads to immune cells. Additionally, the co-location of FITC-OVA and rhodamine-CL264 in immune cells further confirmed the stability of the self-assembled mixed micelles in vivo, since free OVA and CL264 hardly migrated into lymph node and uptake by immune cells without the help of vehicles. These in vivo results were consistent with the stability assay of the mixed micelles in vitro. The selective accumulation of mixed micelles in DCs was then investigated using anti-DC antibody. Around half of CD11c+ cells (DCs) from mice treated with carboxylated-NPs/OVA/CL264 exhibited FITC-signals, much higher than those from hydroxylated-NPs/OVA/CL264 treated mice, 20


Page 20 of 40

Page 21 of 40 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


suggesting

scavenger

receptor-mediated

endocytosis

could

significantly

increase

the

uptake-efficiency of carboxylated-NPs/OVA/CL264 by DCs in LNs, which correlated well with the results of cellular uptake in vitro test.

Fig.5. Carboxylated-NPs enhance antigen and adjuvant accumulation in DLNs after subcutaneous injection. C57BL/6 mice were injected with FITC-OVA and rhodamine-CL264 co-loaded hydroxylated-NPs, Pluronic-NPs, carboxylated-NPs or the physical mixture of OVA+CL264. 3h after administration, inguinal LNs were carefully isolated and the FITC+/rhodamine B+ double positive cells were analyzed by flow cytometry. A) Representative flow cytometry plots of the percentages of FITC+/rhodamine B+ double positive immune cells in DLNs. B) Histogram of the % 21



FITC+/rhodamine B+ double positive cells. To evaluate the above nanovaccines in CD11c+ cells (DC). C) The CD11c+ cells were first gated from ILN cells based on allophycocyanin (APC) fluorescence, and then the uptake of nanovaccines by CD11c+ cells was analyzed based on FITC fluorescence (D). E) Histogram of the percentage of nanovaccines uptake by CD11c+ cells (DC). (Data are presented as mean ± SD n = 5 *P < 0.05 carboxylated-NPs/OVA/CL264, hydroxylated-NPs/OVA/CL264 and Pluronic-NPs/OVA/CL264 vs physical mixture of free OVA+CL264, #P < 0.05 carboxylated-NPs/OVA/CL264 vs hydroxylated-NPs/OVA/CL264 and Pluronic-NPs/OVA/CL264). 3.7 Carboxylated-NPs/OVA/CL264 enhance T cell responses in vivo. To evaluate whether nanovaccines could elicit a potent cellular immunity in vivo, C57BL/6 mice were subcutaneously injected with various formulations of nanovaccine three times on day 0, 7 and 14. Mice were sacrificed one week after the last immunization, and the CD8+ T cell response was determined through ex vivo re-stimulation of isolated splenocytes with 100µg/mL of class I OVA peptide (OVA257−264) and the quantification of IFN-γ producing CD8+ T cells was analyzed by flow cytometry. As shown in Fig.6B, carboxylated-NPs/OVA/CL264 resulted in a high level of IFN-γ+ CD8+ T cells as measured by intracellular cytokine staining (~3.74%), generating a 4.06-fold increase over the physical mixture of free OVA+CL264 (~0.92%). This result suggested that the co-delivery of antigen and immunostimulatory adjuvant via the form of nanoparticles could induce a more potent antigen-specific immune response than their unformulated counterparts. In addition, it was found that carboxylated-NPs/OVA/CL264 were superior to hydroxylated-NPs/OVA/CL264 (~2.46%) or Pluronic-NPs/OVA/CL264 (~1.72%) in the generation of CD8+ T cells responses. This might be due to the synergistic effect of carboxylated-NPs/OVA/CL264 in efficient vaccine delivery and superior antigen presentation pathway. An ideal vaccine could activate CD8+ as well as CD4+ T cells. CD4+ T cells play an important role in initiating adaptive immunity, which then differentiate to subsets of helper T cells such as Th1 cells and Th2 cells. Th1 and Th2 cell subsets are known to promote activation of cytotoxic T lymphocytes (CTLs) or B cells via secretion of distinct patterns of cytokine34, 41. Therefore, whether the carboxylated-NPs/OVA/CL264 could induce the strong antigen-specific CD4+ T cell responses was assessed. We re-stimulated splenocytes ex vivo with 100µg/mL of the 22


Page 22 of 40

Page 23 of 40 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


class II OVA peptide (OVA323 339) and the production of IFN-γ by CD4+ T cells was measured by −

flow cytometry. In accord with the results of CD8+ T cells, shown in Fig.6C, mice immunization with carboxylated-NPs/OVA/CL264 dramatically enhanced the level of IFN-γ+ CD4+ T cells (~2.98%) relative to the other experimental groups. In summary, carboxylated-NPs/OVA/CL264 enhanced both antigen-specific CD4+ T and CD8+ T cell responses, which could trigger a potent cellular and humoral immune response in vivo.

23



Page 24 of 40

Fig.6. Carboxylated-NPs/OVA/CL264 induce potent antigen-specific immune responses in vivo. A) Immunization schedule; mice (n = 5) were immunized on day 0, 7 and 14. B-C) Splenocytes were collected

from

mice

immunized

with

physical

mixture

of

OVA+CL264,

Pluronic-NPs/OVA/CL264, hydroxylated-NPs/OVA/CL264 or carboxylated-NPs/OVA/CL264 and the percentages of IFN-γ+ CD8+ T cells or IFN-γ+ CD4+ T cells after 3d re-stimulation with OVA257−264 or OVA323−339 were measured by flow cytometry. B) Left: Representative flow cytometry plots of the percentages of IFN-γ+ CD8+ T cells. Right: Histogram of the % IFN-γ+ CD8+ T cells. C) Left: Representative flow cytometry plots of the percentages of IFN-γ+ CD4+ T cells. Right: Histogram of the % IFN-γ+ CD4+ T cells. (Data are presented as mean ± SD n = 5 *P

Synthetic polymeric mixed micelles target to lymph node triggering

Recommend Documents