Peptide Amphiphile Micelle Vaccine Size and Charge Influence the

Subscriber access provided by Kaohsiung Medical University

Controlled Release and Delivery Systems

Peptide Amphiphile Micelle Vaccine Size and Charge Influence the Host Antibody Response Rui Zhang, Josiah D. Smith, Brittany N. Allen, Jake S. Kramer, martin schauflinger, and Bret D. Ulery ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00511 • Publication Date (Web): 07 May 2018 Downloaded from http://pubs.acs.org on May 7, 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 29 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 Biomaterials Science & Engineering

Peptide Amphiphile Micelle Vaccine Size and Charge Influence the Host Antibody Response Rui Zhang1, Josiah D. Smith1, Brittany N. Allen2, Jake S. Kramer3, Martin Schauflinger4, and Bret D. Ulery1,2,# 1

Department of Chemical Engineering, University of Missouri, Columbia, Mo 65211

2

Department of Bioengineering, University of Missouri, Columbia, Mo 65211

3

Department of Biochemistry, University of Missouri, Columbia, Mo 65211

4

Electron Microscopy Core Facilities, University of Missouri, Columbia, Mo 65211

#

To whom correspondence should be addressed:

Bret Ulery, Department of Chemical Engineering, W2027 Lafferre Hall, 416 S. 6th Street, University of Missouri 65211. Phone: 573.884.8169. E-mail: [email protected]

KEYWORDS: Immune Modulation; Peptide Amphihile Micelles; Vaccines; Size Effects; Charge Effects

1 ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 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

Abstract

Vaccines are one of the best health care advances ever developed having led to the eradication of small pox and near eradication of polio and diphtheria. While tremendously successful, traditional vaccines (i.e. whole-killed or live-attenuated) have been associated with some undesirable side effects including everything from mild injection site inflammation to the autoimmune disease Guillain-Barre Syndrome. This has led recent research to focus on developing subunit vaccines (i.e. protein, peptide, or DNA vaccines) since they are inherently safer due to delivering only the bioactive components necessary (i.e. antigens) to produce a protective immune response against the pathogen of interest. However, a major challenge in developing subunit vaccines is overcoming numerous biological barriers to effectively deliver antigen to the secondary lymphoid organs where adaptive immune responses are orchestrated. Peptide amphiphile micelles are a class of biomaterials that have been shown to possess potent self-adjuvanting vaccine properties, but their optimization capacity and underlying immunostimulatory mechanism are not well understood. Our enclosed work investigated the influence that micelle size and charge have on materials bioactivity including lymph node accumulation, cell uptake ability, and immunogenicity. The results generated provide considerable insight into how micelles exert their biological effects yielding a micellar toolbox that can be exploited to either enhance or diminish host immune responses. This exciting development makes peptide amphiphile micelles an attractive candidate for both immune activation or suppression applications.


Page 2 of 29

Page 3 of 29 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 Vaccines have played an essential role in preventing disease and greatly decreasing human mortality.1-4 The most commonly utilized vaccine formulations to date consist of either killed or inactivated whole pathogens. Despite the considerable benefits achieved with these vaccines, they can be associated with an array of deleterious drawbacks such as toxicity, arduous production process requirements, and cold chain storage maintenance constraints.5-8 Therefore, as novel vaccine formulations emerge, traditional whole pathogen vaccines have become less appealing.9-11 Rapid growth in immunology and bioinformatics have helped researchers identify the short peptide sequences from whole pathogen vaccines for which vaccine-induced host immune responses are directed.12-15 Creating vaccine formulations comprised of just these immunogenic epitopes is an attractive alternative to whole in-tact vaccines since it eliminates the inclusion of unnecessary pathogenic components and allows for more desirable production and storage options. However, the smaller size and low retention time of peptide epitopes compared to in-tact vaccines are significant factors that negatively impact their functionality.16-19 Thus, an effective peptide delivery vehicle is needed to solve this fundamental issue. Peptide amphiphiles (PAs) are a class of biomaterials comprised of peptide-lipid conjugates that self-assemble into micelles in water. Previous research has demonstrated that peptide amphiphile micelles (PAMs) have the ability to deliver biologically active peptides for a variety of biomedical applications including cancer therapy,16,

18

tissue regeneration,20-22 and disease

diagnostics.23-24 Also, PAMs are comprised of a high concentration of peptide, possess significant peptide enzymatic resistance, and can greatly enhance peptide intracellular 3 ACS Paragon Plus Environment


delivery.25-27 More recently, PAMs have emerged as a promising self-adjuvanting vaccine carrier capable of inducing strong and durable prophylactic antibody-mediated responses.17, 28 While exciting, these preliminary results have yet to establish systematic PAM vaccine design rules. In this study, a well characterized linked-recognition B and T cell peptide epitope sequence, ESLKISQAVHAAHAEINEAGRE (OVABT), was utilized to explore how the physical properties of shape and charge influence PAM immunogenicity. PAMs consisting of four previously designed shapes29 and varying surface charge were explored. Interestingly, the results generated show that micellar physical properties greatly influence host immune responses to peptide antigens allowing for significant potentiation or diminution. These results provide significant insight into the platform technology potential of PAMs allowing for their design not only for vaccine applications, but also for the treatment of cancer and autoimmune diseases.


Page 4 of 29

Page 5 of 29 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


Scheme 1. Four different PAs with the same antigen (OVABT) were designed. Lipid tail number and zwitterion-like block position were modified to yield different PA chemistries.

Results and Discussion PAM size significantly influences its immunogenicity. Previous results from our research group have shown that the inclusion of a zwitterion-like region (i.e. (KE)4) into traditional diblock PAs yields interesting triblock PAs (Scheme 1) with similar surface charge (Figure S1).29 These unique molecules cooperatively undergo hydrophobically-driven self-assembly and electrostatically-driven aggregation yielding PAMs of different shapes and sizes independent of the application-specific peptide sequence.29 Specifically, double-lipid PAs (i.e. Palm2K-OVABT(KE)4 and Palm2K-(EK)4-OVABT) formed small PAMs (i.e. spheres / short cylinders and clusters, respectively) and single-lipid PAs (i.e. PalmK-OVABT-(KE)4 and PalmK-(EK)4OVABT) formed large PAMs (i.e. twines and braids, respectively) as shown in Figure 1a and Table S1.29 To investigate the influence PAM shape, and therefore size, has on their potential to function as self-adjuvanting vaccine carriers, mice were immunized subcutaneously in the nape of the neck with peptide antigen (i.e. OVABT-(KE4)) alone or one of the four PAM formulations for which OVABT-specific IgG antibody production was assessed (Figure 1b). Overall, smaller PAMs were found to facilitate higher IgG antibody production than larger PAMs. In specific, PAM spheres / short cylinders induced significantly higher IgG titers than both PAM twines and braids. PAM clusters, though less immunogenic than PAM spheres / short cylinders, induced slightly and significantly higher IgG titers than PAM twines and braids, respectively. Interestingly, no detectable IgG production was seen in any of animals immunized with PAM 5 ACS Paragon Plus Environment


braids. When compared to vaccination with the peptide antigen alone, PAM size was found to either dramatically enhance (i.e. PAM spheres / short cylinders) or significantly decrease (i.e. PAM braids) antigen-specific antibody responses.

Figure 1. PAM size influences immunogenicity. (a) Negative-stain transmission electron microscopy revealed that tail number and zwitterion-like block position impact PAM nanoarchitecture allowing for the fabrication of spheres / short cylinders (i.e. Palm2K-OVABT(KE)4), clusters (i.e. Palm2K-(EK)4-OVABT), twines (i.e. PalmK-OVABT-(KE)4), and braids (i.e. PalmK-(EK)4-OVABT). (b) Serum was collected from mice vaccinated with OVABT-(KE)4 peptide and the four different PAM formulations two weeks post-boost immunization. Total IgG


Page 6 of 29

Page 7 of 29 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


titers measured by ELISA revealed that PAM size significantly influenced the immunogenicity of the incorporated peptide antigen (i.e. OVABT-(KE)4). Within the graph, groups that possess different letters have statistically significant differences in mean (p ≤ 0.05) whereas those that possess the same letter are similar (p > 0.05) PAM size considerably influences in vivo trafficking and cellular uptake. To probe how PAM size augments host immune responses against PAM incorporated antigen, further biological experiments were conducted. As lymph nodes are the principal organs where antibody production is coordinated, PAMs must deliver their antigenic payload there to facilitate the desired adaptive immune response. Followed by interstitial injection, antigens can reach lymph nodes via two different pathways, direct drainage or transport by tissue residential antigen presenting cells (APCs) at the injection site.11 Recent work has demonstrated that direct drainage is the dominant pathway specifically for subcutaneously administrated micelle vaccines as lymph node accumulation was observed within 1 hour post-vaccination.17 Therefore, after PAMs reach the lymph nodes, they will need to be appropriately processed by APCs such as macrophages (MØs) and dendritic cells (DCs) which play a crucial role in initiating T cell and B cell mediated immune responses. Mice were injected similarly to the vaccination study with one of the four PAM formulations, each fluorescently-labeled so their in vivo trafficking behavior could be monitored. At 24 hours post-delivery, the injection site draining lymph nodes (i.e. axillary and brachial) were harvested and fluorescence was visualized by an in vivo imaging system (Figure S2) which was standardized to determine differences among PAM formulations (Figure 2a). Interestingly, peptide and PAM braids showed no detectable accumulation in the draining lymph nodes as compared to significant accumulation of PAM spheres / short cylinders, clusters, and twines. As the vaccine formulations span several orders of magnitude in size from nanometers 7 ACS Paragon Plus Environment


(i.e. peptide) to tens of nanometers (i.e. PAM spheres / short cylinders, Table S1) to hundreds of nanometers (i.e. PAM clusters, Table S1), to microns (i.e. PAM twines) to tens of microns (i.e. PAM braids),29 there appears to be an effective range over which PAMs can readily traffic to the draining lymph node which corresponds to previously published work.30-31 In order to assess the internalization capacity of PAMs with different size, fluorophore-labeled formulations were incubated with MØs for 1 hour after which their cell association was evaluated by confocal microscopy (Figure 2b) and flow cytometry (Figure S3). In specific, peptide, PAM twines, and PAM braids were all found to be minimally associated with MØs whereas PAM spheres / short cylinders and PAM clusters showed significant cell association over background. Interestingly, though four times the amount of PAM clusters were found associated with MØs compared to PAM spheres / short cylinders (Figure S3), approximately four times more were found associated with the cell membrane instead of internally trafficked (Table S2). Thus, this sustained membrane co-localization may result in slightly decreased immunogenicity for PAM clusters compared to PAM spheres / short cylinders. DCs showed similar PAM size-dependent association (Figure S4, Figure S5, and Table S3).


Page 8 of 29

Page 9 of 29 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 2. PAM size influences lymph node accumulation and cell association. (a) Draining lymph nodes were collected 24 hours after immunization and fluorescence was quantified and standardized by IVIS. Less vaccine accumulated in lymph nodes when their size is either very small (i.e. peptide - ~ 1 - 3 nm) or quite large (i.e. PAM braids - ~ 10 µm or more). (b) MØs were incubated with different vaccine formulations for 1 hour, followed by fixing and mounting on coverslips, before being assessed by confocal fluorescent microscopy. Vaccine formulations tens to hundreds of nanometers in size (i.e. PAM spheres / short cylinders and clusters) were more readily associated with cells than smaller (i.e. peptide) or larger (i.e. PAM twines or braids) products. Within the graph, groups that possess different letters have statistically significant differences in mean (p ≤ 0.05) whereas those that possess the same letter are similar (p > 0.05). MØ uptake of different PAMs were further assessed by transmission electron microscopy (TEM) coupled with high pressure freezing (HPF) (Figure 3 and Figure S6). This allowed us to evaluate initial interactions between PAMs and cells at a very early time point (i.e. 15 mins). Results indicated that the four different PAMs were likely intact, or at least not appreciably disassociated, when they were internalized by MØs. Interestingly, this result is dissimilar to previously published results that support the concept that PAMs dissociate into individual amphiphiles that insert into the outer leaflet of the lipid bilayer facilitating their internalization.3234

These papers utilized cancer cells, T lymphocytes, fibroblasts, myeloblasts, and hematopoietic

cells, all of which are expected to have minimal turnover of their lipid bilayers. In contrast, MØs are phagocytic cells which constantly renew their cell membranes making it possible that PAMs did not have sufficient contact time with the lipid bilayer to disassociate into individual amphiphiles before being internalized. Previous research has demonstrated that chemically tethering lipids to hydrophilic molecules (e.g. peptides and DNA) to form amphiphilic 9 ACS Paragon Plus Environment


biomaterials (e.g. peptide amphiphile and DNA amphiphile) can greatly improve their cell uptake.27, 35 However, we did not observe improved internalization of single tail PAMs (i.e. PAM twines and PAM braids) by MØs (Figure 2b). This result may arise from cells attempting to internalize intact PAMs allowing for large PAMs to inhibit their uptake.

Figure 3. Phagocytic cells internalize intact PAMs. MØs were incubated with the four different PAM formulations (i.e. (a) PAM spheres / short cylinders, (b) PAM clusters, (c) PAM twines, and (d) PAM braids) for 15 mins before high presssure freezing was employed. Thin sections (75 nm) were prepared and mounted on the formvar/carbon coated grids before being viewed by transmission electron microscopy. The micrographs provide significant evidence that all four different PAMs stayed intact during the cell internalization process. PAMs are designated by the red arrows. Peptide terminus modification can sometimes alter processed antigen MHC I presentation, but is less concerning for MHC II presentation, especially the OVABT sequence.36 This is one of the reason that OVAun modifications have been so widely studied.37-40 Therefore, although OVABT antigen within PAMs is linked by different flanking regions, it unlikely to be a factor contributing to the varied immunogenicity observed for the different formulations. Hence, the


Page 10 of 29

Page 11 of 29 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


size of different PAMs, rather than their chemical structure, is believed to play the dominant role in affecting their ability to induce a host antibody response. PAM charge impacts immunogenicity. The surface charge of vaccines plays a critical role in determining their immunogenicity, cytotoxicity, and payload (e.g. nucleic acid or protein).41-44 In many cases, highly positively-charged vaccines exert significant cytotoxicity and off-target effects45-46 whereas highly negatively-charged vaccines often possess limited cell uptake ability.47-48 Therefore, care must be taken to utilize the optimal surface charge for when designing PAM vaccines. The results above demonstrate that PAM spheres / short cylinders (i.e. Pam2K-OVABT-(KE)4) are the optimal size to enhance peptide immunogenicity. Thus, we chose this formulation to investigate the influence PAM vaccine charge has on materials immunogenicity. PAM vaccine charge modification was achieved by altering the neutral zwitterion-like hydrophilic region (i.e. (KE)4) to either a positively-charged region (i.e. K8) or a negatively-charged region (i.e. E8). These sequences yield zwitterion-like PAMs (Palm2KOVABT-(KE)4), cationic PAMs (Palm2K-OVABT-K8), or anionic PAMs (Palm2K-OVABT-E8) (Scheme 2). Micelle surface charge was assessed via zeta potential measurement for which zwitterion-like PAMs were found to have a moderate positive charge compared to the higher positive charge seen with cationic PAMs and the strong negative charge observed with anionic PAMs (Figure 4a).



Scheme 2. PAM surface charge is readily tunable. (a) PAM surface charge modification was achieved by altering the zwitterion-like region (i.e. (KE)4) to be either cationic (i.e. K8) or anionic (i.e. E8). This allowed for the fabrication of zwitterion-like PAMs (i.e. Palm2K-OVABT(KE)4), cationic PAMs (i.e. Palm2K-OVABT-K8), and anionic PAMs (i.e. Palm2K-OVABT-E8). To assess the influence PAM charge has on their potential to function as vaccine carriers, potential size differences needed to be excluded as a possible confounding variable. Negativestain TEM and dynamic light scattering revealed that the three different PAMs all possessed similar size and structure (i.e. spheres or short cylinders) (Figure 4b and Table S4). The immunogenicity of the three different PAMs was evaluated similarly to before employing subcutaneous vaccination and antigen-specific IgG characterization. The results indicated that both cationic PAMs and anionic PAMs induced significantly lower IgG titers than zwitterionlike PAMs (Figure 4c). Specifically, cationic PAMs still induced some level of antibody


Page 12 of 29

Page 13 of 29 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


production while anionic PAMs diminished the antigen-specific antibody response to subdetection levels.



Figure 4. PAM charge influences immunogenicity. (a) PAM surface charge was evaluated by zeta potential measurement revealing that cationic PAMs and anionic PAMs were significantly more positively and negatively charged, respectively, than zwitterion-like PAMs. (b) Negativestain transmission electron microscopy revealed that zwitterion-like, cationic, and anionic PAMs all possess similar shape and size. (c) Serum was collected from mice vaccinated with the three different PAM formulations two weeks post-boost immunization. Total IgG titers measured by ELISA revealed that PAM charge significantly modulated the host immune response to the micelle incorporated peptide antigen (i.e. OVABT-(KE)4). Within the graph, groups that possess different letters have statistically significant differences in mean (p ≤ 0.05) whereas those that possess the same letter are similar (p > 0.05). PAM charge significantly affects in vivo trafficking and cell uptake Similar to the more in-depth analysis conducted to probe vaccine size, further biological experiments were carried out to explore the influence of vaccine charge. To assess the lymph node draining ability of the differently charged PAMs, mice were injected similarly to the vaccination study with fluorophore-tagged PAMs. At 24 hours post-vaccination, both axillary and brachial lymph nodes were harvested, fluorescence was visualized by IVIS (Figure S7) and standardized for comparison (Figure 5a). Interestingly, cationic PAMs had significantly lower accumulation in the lymph nodes than both zwitterion-like PAMs and anionic PAMs. This difference is presumed to be caused by non-specific association between positively-charged micelles and the negatively-charged lipid bilayer of any of the cells that would be encountered on the way to the lymph nodes. This hypothesis is supported by previously reported similar observations with other cationic delivery devices.49-51 14 ACS Paragon Plus Environment

Page 14 of 29

Page 15 of 29 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 assess the internalization capacity of PAMs with different charge, fluorophore-tagged formulations were cultured with MØs for 1 hour. Micelle-cell association was subsequently characterized by confocal microscopy (Figure 5b) and flow cytometry (Figure S8). Cationic PAMs unsurprisingly had slightly higher cell association than zwitterion-like PAMs. While this is beneficial from an APC processing standpoint, positive surface charge also increased cationic PAM off-target effects as these micelles had increased non-phagocytic cell association compared to zwitterion-like PAMs (Figure S9). On the other hand, anionic PAMs showed significantly weaker cell association than zwitterion-like PAMs or cationic PAMs. This is likely due to the electrostatic repulsion force between the anionic region (E8) and the negatively charged cell lipid bilayer. Thus, though anionic PAMs, like some other anionic materials, can reach lymph nodes without issue and temporarily reside in the tissue,52-53 their weak ability to interact with cells may result in their diminished immunogenicity. DCs showed similar PAM charge-dependent association (Figure S10 and Figure S11). Therefore, micelle surface charge skewing too positive or too negative can adversely affect the capacity for PAMs to function as a selfadjuvanting vaccine delivery system.



Figure 5. PAM charge influences lymph node accumulation and cell association. (a) Draining lymph nodes were harvested 24 hours after immunization for which fluorescence was determined by IVIS and standardized for comparison. Cationic PAMs showed less micelle accumulation than zwitterion-like PAMs or anionic PAMs. (b) MØs were incubated with the three different PAM formulations for 1 hour. After fixation and mounting on coverslips, samples were evaluated via confocal fluorescent microscopy. Anionic PAMs showed reduced cell uptake than the other two formulations tested. Taken together, highly charged micelles inhibit the in vivo trafficking or cellular processing vaccines require to initiate a productive adaptive immune response. Within the graph, groups that possess different letters have statistically significant differences in mean (p ≤ 0.05) whereas those that possess the same letter are similar (p > 0.05) Conclusion


Page 16 of 29

Page 17 of 29 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


The data presented provides additional support to the well-established theory that vaccine immunogenicity is most significantly governed by two important factors: the capacity to accumulate in the lymph nodes and the ability to interact properly with APCs.54 These criteria are deceptively straightforward as numerous biological barriers actually exist between injecting a vaccine and having it properly deliver antigenic payload in a manner that induces productive adaptive host immune responses. Therefore, when trying to enhance the bioactivity of weak immunogens like subunit vaccines, special care must be taken to properly engineer the delivery vehicle. Carrier size and charge have been found to play important roles in modulating vaccine payload immunogenicity. Specifically, lymph node draining ability and cell uptake capacity have been found to be size-dependent with vehicles ~ 10 – 200 nm in diameter tending to be the most immunogenic.30, 55-57 This behavior has been shown to be relatively independent of materials chemistry, since vehicles in this size range can effectively enter lymphoid vessels while also being able to be efficiently internalized by APCs.31 Where chemistry has been found to play a role is when it influences charge as this can greatly impact cell uptake ability and off-target effects.47, 58 Since the lipid bilayer of cells is quite negatively charged due to its rich phosphate content, vaccine carrier charge influences the likelihood of repulsive, neutral, or attractive interactions with cells. Highly positively charged molecules (e.g. cell penetrating peptides, cationic lipids, and cationic polymers) can facilitate greatly enhanced cell uptake because of electrostatic attraction, but are often accompanied by deleterious effects such as off-target association and significant toxicity.59-62 On the other hand, highly negatively charged molecules may create electrostatic repulsion with the lipid bilayer inhibiting their internalization.47



Therefore, neutral or modest surface charge is preferred in order to facilitate appropriate APC association and internalization. Our experimental results agree with conclusions from many other researchers as PAMs tens to hundreds of nanometers in size with moderately positive surface charge were capable of facilitating the strongest enhancement of host antibody production to the micelle-associated antigen. The Palm2K-Peptide-(KE)4 formulation that yields these micelles is thus a promising vaccine carrier candidate for which a variety of peptide antigens can be readily inserted. Additionally, PAM braids, though not be a suitable candidate for vaccination, hold tremendous potential for other biomedical applications. The capacity to utilize the PalmK-(EK)4-Peptide formulation to specifically prevent the induction of adaptive immune responses to an included peptide could enhance tissue regeneration22, 63-66 or anti-inflammatory responses.67 The presented work combined with our previous results highlighting the considerable synthetic flexibility of PAMs29 provides significant evidence that these self-assembled biomolecular materials can function as a platform technology that can be readily engineered to address a variety of biomedical challenges.

Methods and Materials Peptide and peptide amphiphile synthesis and purification. Side chain protected peptides ((EK)4-OVABT and OVABT-(KE)4) on rink amide resin were purchased from Synpeptide Co., Ltd (Shanghai, China). The N-terminal Fmoc group was removed by treatment with 25% piperidine in dimethylformamide (DMF) for 15 min. Control peptide products (i.e. OVABT-(KE)4 and FAM18 ACS Paragon Plus Environment

Page 18 of 29

Page 19 of 29 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


labeled OVABT-(KE)4) were created by N-terminal Fmoc deprotection followed by acetylation with significant excess of acetic anhydride or fluorophore attachment using 5 eq. 5,6carbonxyfluorescein (FAM), 4.2 eq. HBTU, 5 eq. HOBT, and 10 eq. DIEA. For peptide amphiphile (PA) synthesis, a non-native lysine was then attached to allow for the addition of palmitic acid(s) (Palm). Either Fmoc-Lys(ivDde)-OH or Fmoc-Lys(Fmoc)-OH was used with orthogonal deprotection to create single-lipid (i.e. PalmK-(EK)4-OVABT and PalmK-OVABT(KE)4) or double-lipid (i.e. Palm2K-(EK)4-OVABT and Palm2K-OVABT-(KE)4) PAs, respectively. ivDde was removed by treating the peptide on resin with 2% Hydrazine in DMF whereas Fmoc removal was facilitated by 25% piperidine in DMF. Lipid conjugation was carried out using 5 eq. palmitic acid, 4.2 eq. HBTU, 5 eq. HOBT, and 10 eq. DIEA. For single-lipid PAs, the Nterminus was acetylated with a significant excess of acetic anhydride. Fluorophore-labeled, single-lipid PAs were synthesized by using ivDde-Lys(Fmoc) instead of the previously mentioned Fmoc-Lys(ivDde). This allowed for ivDde deprotection and FAM attachment on the N-terminus of the PA after lipid conjugation. FAM-labeled, double-lipid PAs were synthesized by first adding a second non-native lysine (i.e. Fmoc-Lys(ivDDE)-OH) between the previously mentioned Fmoc-Lys(Fmoc) and the N-terminus of the peptide on resin for which ivDde deprotection and FAM conjugation were carried out after lipid attachment. All on-resin reactions were conducted manually in a glass reaction vessel (Chemglass, Vineland, NJ). All peptides and PAs were cleaved from resin and their side groups deprotected via a single-step reaction consisting of 2 h exposure to the following mixture: TFA, thioanisole, phenol, water, ethandithiol and triisopropylsilane (87.5:2.5:2.5:2.5:2.5). Precipitation and multiple washes with diethyl ether yielded crude peptide or PA. All products synthesized were characterized by analytical high-pressure liquid chromatography (HPLC, Beckmann Coulter, Fullerton, CA) and 19 ACS Paragon Plus Environment


purified by mass spectrometry aided semi-preparative high-pressure liquid chromatography (LCMS) using either a C4 or C18 column (Milford, MA) and in-house optimized solvent gradients. Micelle characterization. Micelle morphology was assessed by negative stain transmission electron microscopy (TEM). Product solutions (5 µL of 100 µM) were added to glow-discharged, carbon support TEM grids (200 mesh, Electron Microscopy Sciences) and incubated for 5 mins. Filter paper was used to wick away excess solution and 5 µL of nanotungsten (Nanoprobes, Inc) was immediately added. After 5 mins of incubation, grids were blotted dry and imaged with a JEOL JEM-1400 TEM at 120 kV. Zeta potential and dynamic light scattering measurements were obtained in triplicate using a Zetasizer NanoZ for which Huckel model and backscattering mode were used respectively to fit the data. For each sample (1 mL of 5 µM), 20 runs were performed using the auto-analysis mode. In vivo immunization studies. All in vivo experiments were performed under approval from the Animal Care and Use Committee (ACUC) at the University of Missouri. Sex-matched Balb/C mice (4 males and 4 females per group) were obtained from Jackson Laboratories and subcutaneously administrated different vaccine formulations in the nape of the neck. Primary injections consisted of 200 nmol of peptide vaccine or one of the PAM vaccines in 100 µL of phosphate buffered saline (PBS). Boost injections consisting of half the primary dose (100 nmol vaccine in 50ml of PBS) were given 4 weeks later. Whole blood was collected from the saphenous vein 2 week after boost injection (i.e. 6 weeks post-primary injection) and centrifuged at 10,000 rpm for 10 min to separate out the red blood cells. The resulting supernatant serum was harvested and stored at −80 °C until further analysis.


Page 20 of 29

Page 21 of 29 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


Antibody titer assessment. High binding, 96-well ELISA plates (Santa Cruz Biotechnology) were coated overnight with 4 µg/mL OVABT peptide (Ac-ESLKISQAVHAAHAEINEAGRENH2) in PBS. Wells were washed with PBS-T (0.05% Tween-20 in PBS) and blocked with 10% fetal bovine serum (FBS) in PBS (blocking buffer) for 1 hour. Serum was serially diluted twofold in blocking buffer across the plate and incubated for 2 hours. Wells were then washed with PBS-T and incubated with 1:3000 diluted detection antibody for 1 hour. After additional washing with PBS-T, wells were incubated for 30 mins with 100 µL TMB substrate (Biolegend) and optical density (OD) was measured at 650 nm absorbance using a Biotek Cytation 5 spectrofluorometer. End-point antibody titers were defined as the greatest serum dilution where OD was at least twice that of serum from mice vaccinated with PBS. If end-point titers were not reached with one plate, then additional titrations were utilized until ODs were diluted below detection. Lymph node (LN) imaging. Jackson Laboratories sex-matched Balb/C mice (3 males and 3 females per group) were subcutaneously administrated different fluorescent vaccine formulation in the nape of neck. Injections consisted of 200 nmol of vaccines (20% FAM labeled and 80% unlabeled) in 100 µL of PBS. Animals were sacrificed 24 hours after injection and axillary lymph nodes were excised and fluorescently imaged using a Xenogen IVIS 200 (PerkinElmer, Waltham, MA). Quantification of the fluorescence signal was achieved using Living Image software. LN fluorescence was standardized based on the inherent vaccine fluorescence intensity as measured by the same equipment. Preparation of bone marrow-derived dendritic cells (DCs). Balb/c mouse femurs and tibias were harvested from which cells were collected by flushing the bone marrow with complete 21 ACS Paragon Plus Environment


RPMI 1640 media that was passed through a cell strainer (70 µM mesh size). Red blood cells were lysed by ammonium-chloride-potassium (ACK) lysis buffer before stromal cells were seeded on non-tissue culture treated petri-dishes. The cells were cultured in BMDC differentiation media (RPMI 1640 supplemented with 10% fetal bovine serum - FBS, 1% penicillin-streptomycin, 50 µM β-mercaptoethanol, and 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF)) at 37 °C with 5% CO2 for which culture media was refreshed on days 3, 6, and 8. Any stromal cells expected to have differentiated into BMDCs should have done so by 10 days of incubation, so the mixed cell population was purified using mouse CD11c nano beads (Biolegend) at that time point. Cell uptake assessment. RAW 264.7 (macrophage-like cells - MØs) or DCs were seeded at a density of 2 x 105 cells/well on coverslips in a 24-well plate and incubated overnight. After 1 hour incubation with 5 µM FAM-labeled PAMs, cells were washed with PBS and fixed with 4% paraformaldehyde. The cell membrane was stained with CF594-labeled wheat germ agglutinin (CF594-WGA) after which coverslips were then mounted on a microscope slide with DAPI (4’,6-diamidino-2-phenylindole) containing mounting media for at least 24 hours before being viewed with a confocal fluorescent microscope (Leica TCS SP8). For flow cytometry, a similar protocol was followed except MØs, DCs, or naïve T cells (isolated from lymph nodes using a MojoSort Mouse CD4 Naïve T Cell isolation kit) were seeded at a density of 2 x 105 cells/well in a 24-well plate and incubated overnight. MØs were incubated with 5 µM FAM-labeled PAMs for 1 hour at 37 °C with 5% CO2 followed by washing with PBS. Cells were then fixed with 4% paraformaldehyde and analyzed by a flow cytometer (BD LSRFortessa X20). Standardized MFI was calculated based on inherent vaccine fluorescence intensity as measured by the previously mentioned spectrofluorometer. 22 ACS Paragon Plus Environment

Page 22 of 29

Page 23 of 29 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


High pressure freezing (HPF). MØs were first grown on gold-coated sapphire discs (3 mm diameter; Wohlwend GmbH, Switzerland). To increase events resolution, a higher concentration of PAMs (100 µM) were incubated with the MØs and processed after 15 mins to prevent lysosomal degradation. The MØs were then cryo-immobilized by high-pressure freezing using a Wohlwend HPF Compact 02. Super quick freeze substitution was performed over a period of 2.5 h.68 A freeze substitution cocktail consisting of acetone with 0.5% (w/v) osmium tetroxide, 0.1% (w/v) uranyl acetate, 0.1% (w/v) imidazole, and 4% (v/v) water was added to mitigate the process. Samples were infiltrated with Epon/Araldite and 75 nm thin sections were prepared and mounted on formvar/carbon coated slot grids. Samples were imaged with a JEOL JEM-1400 transmission electron microscope at an acceleration voltage of 80 kV. Statistical analysis. JMP software (SAS Institute) was used to make comparisons between groups where an analysis of variance (ANOVA) was performed followed by Tukey’s HSD testing to determine pairwise statistically significant differences (p < 0.05). Within graphs, groups that possess different letters have statistically significant differences in mean whereas those that possess the same letter are statistically insignificant.

Associated Content Author Information. Corresponding Author – Bret Ulery; [email protected]; 573-884-8169 Author Contributions. R.Z. and B.D.U. designed the experiments. R.Z. carried out materials synthesis and characterization with M.S. preparing the high pressure freezing microscopy grids. R.Z., J.D.S., and B.N.A. conducted the animal experiments and R.Z. and J.S.K. assessed 23 ACS Paragon Plus Environment


antibody titers. R.Z. performed the in vitro cell experiments. B.D.U. supervised the experiments and R.Z. and B.D.U wrote the manuscript. Funding Sources. This work was supported by start-up funding from the University of Missouri as well as grants from the University of Missouri Electron Microscopy Core, the University of Missouri Research Board, and the PhRMA Foundation. Acknowledgements. We thank Professor Thomas Phillips, Professor Jeffrey Adamovicz, and Ms. Alexis Dadelahi for useful discussions on the enclosed data and manuscript. We also thank Dr. Fabio Gallazzi in the Molecular Interactions Core at the University of Missouri for purifying the peptides and peptide amphiphiles used in this research. Additionally, we thank Ms. Ashley Berendzen for her assistance in organ imaging. Supporting Information. Additional tables and figures including dynamic light scattering, zeta potential, transmission electron micrographs, and dendritic cell internalization assessment are free of charge on the ACS publication website.

References 1.

2.

3. 4.

Roush, S. W.; Murphy, T. V.; Group, V.-P. D. T. W., Historical comparisons of morbidity and mortality for vaccine-preventable diseases in the United States. Jama 2007, 298 (18), 2155-2163. Fine, P. E.; Carneiro, I. A., Transmissibility and persistence of oral polio vaccine viruses: implications for the global poliomyelitis eradication initiative. American journal of epidemiology 1999, 150 (10), 1001-1021. Kennedy, R. B.; Ovsyannikova, I. G.; Jacobson, R. M.; Poland, G. A., The immunology of smallpox vaccines. Current opinion in immunology 2009, 21 (3), 314-320. Organization, W. H., Hepatitis B vaccines. Weekly epidemiological record 2009, 84 (40), 405-419. 24 ACS Paragon Plus Environment

Page 24 of 29

Page 25 of 29 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


5.

6. 7.

8. 9. 10. 11. 12.

13.

14.

15.

16.

17.

18. 19. 20.

21.

Berry, N.; Davis, C.; Jenkins, A.; Wood, D.; Minor, P.; Schild, G.; Bottiger, M.; Holmes, H.; Almond, N., Vaccine safety: Analysis of oral polio vaccine CHAT stocks. Nature 2001, 410 (6832), 1046-1047. Chen, R. T., Vaccine risks: real, perceived and unknown. Vaccine 1999, 17, S41-S46. Matthias, D. M.; Robertson, J.; Garrison, M. M.; Newland, S.; Nelson, C., Freezing temperatures in the vaccine cold chain: a systematic literature review. Vaccine 2007, 25 (20), 3980-3986. Techathawat, S.; Varinsathien, P.; Rasdjarmrearnsook, A.; Tharmaphornpilas, P., Exposure to heat and freezing in the vaccine cold chain in Thailand. Vaccine 2007, 25 (7), 1328-1333. Babiuk, L. A., Broadening the approaches to developing more effective vaccines. Vaccine 1999, 17 (13), 1587-1595. Brown, F., Peptide vaccines: fantasy or reality? World Journal of Microbiology and Biotechnology 1992, 8, 52-53. Levine, M. M.; Sztein, M. B., Vaccine development strategies for improving immunization: the role of modern immunology. Nature immunology 2004, 5 (5), 460-464. Zhang, C.; Zheng, G.; Xu, S.-F.; Xu, D., Computational challenges in characterization of bacteria and bacteria-host interactions based on genomic data. Journal of Computer Science and Technology 2012, 27 (2), 225-239. Wan, X.-F.; Ataman, D.; Xu, D., Application of computational biology in understanding emerging infectious diseases: inferring biological function for SM complex of SARS-CoV. New York, Nova Science Publishers: 2005. Moutaftsi, M.; Peters, B.; Pasquetto, V.; Tscharke, D. C.; Sidney, J.; Bui, H.-H.; Grey, H.; Sette, A., A consensus epitope prediction approach identifies the breadth of murine TCD8+cell responses to vaccinia virus. Nature biotechnology 2006, 24 (7), 817-819. Saha, S.; Raghava, G., Prediction of continuous B‐cell epitopes in an antigen using recurrent neural network. Proteins: Structure, Function, and Bioinformatics 2006, 65 (1), 40-48. Black, M.; Trent, A.; Kostenko, Y.; Lee, J. S.; Olive, C.; Tirrell, M., Self‐Assembled Peptide Amphiphile Micelles Containing a Cytotoxic T‐Cell Epitope Promote a Protective Immune Response In Vivo. Advanced Materials 2012, 24 (28), 3845-3849. Barrett, J. C.; Ulery, B. D.; Trent, A.; Liang, S.; David, N. A.; Tirrell, M., Modular peptide amphiphile micelles improve an antibody-mediated immune response to Group A Streptococcus. ACS Biomaterials Science & Engineering 2016, 3 (2), 144-152. Trent, A.; Marullo, R.; Lin, B.; Black, M.; Tirrell, M., Structural properties of soluble peptide amphiphile micelles. Soft Matter 2011, 7 (20), 9572-9582. Zhang, R.; Ulery, B. D., Synthetic Vaccine Characterization and Design. Journal of Bionanoscience 2018, 12 (1), 1-11. Webber, M. J.; Tongers, J.; Newcomb, C. J.; Marquardt, K.-T.; Bauersachs, J.; Losordo, D. W.; Stupp, S. I., Supramolecular nanostructures that mimic VEGF as a strategy for ischemic tissue repair. Proceedings of the National Academy of Sciences 2011, 108 (33), 1343813443. Anderson, J. M.; Kushwaha, M.; Tambralli, A.; Bellis, S. L.; Camata, R. P.; Jun, H.-W., Osteogenic differentiation of human mesenchymal stem cells directed by extracellular matrix-mimicking ligands in a biomimetic self-assembled peptide amphiphile nanomatrix. Biomacromolecules 2009, 10 (10), 2935-2944. 25 ACS Paragon Plus Environment


22. Mata, A.; Geng, Y.; Henrikson, K. J.; Aparicio, C.; Stock, S. R.; Satcher, R. L.; Stupp, S. I., Bone regeneration mediated by biomimetic mineralization of a nanofiber matrix. Biomaterials 2010, 31 (23), 6004-6012. 23. Mlinar, L. B.; Chung, E. J.; Wonder, E. A.; Tirrell, M., Active targeting of early and midstage atherosclerotic plaques using self-assembled peptide amphiphile micelles. Biomaterials 2014, 35 (30), 8678-8686. 24. Chung, E. J.; Mlinar, L. B.; Nord, K.; Sugimoto, M. J.; Wonder, E.; Alenghat, F. J.; Fang, Y.; Tirrell, M., Monocyte‐Targeting Supramolecular Micellar Assemblies: A Molecular Diagnostic Tool for Atherosclerosis. Advanced healthcare materials 2015, 4 (3), 367-376. 25. Peters, D.; Kastantin, M.; Kotamraju, V. R.; Karmali, P. P.; Gujraty, K.; Tirrell, M.; Ruoslahti, E., Targeting atherosclerosis by using modular, multifunctional micelles. Proceedings of the National Academy of Sciences 2009, 106 (24), 9815-9819. 26. Chung, E. J.; Mlinar, L. B.; Sugimoto, M. J.; Nord, K.; Roman, B. B.; Tirrell, M., In vivo biodistribution and clearance of peptide amphiphile micelles. Nanomedicine: Nanotechnology, Biology and Medicine 2015, 11 (2), 479-487. 27. Missirlis, D.; Teesalu, T.; Black, M.; Tirrell, M., The non-peptidic part determines the internalization mechanism and intracellular trafficking of peptide amphiphiles. PloS one 2013, 8 (1), e54611. 28. Trent, A.; Ulery, B. D.; Black, M. J.; Barrett, J. C.; Liang, S.; Kostenko, Y.; David, N. A.; Tirrell, M. V., Peptide amphiphile micelles self-adjuvant group A streptococcal vaccination. The AAPS journal 2015, 17 (2), 380-388. 29. Zhang, R.; Morton, L. D.; Smith, J. D.; Gallazzi, F.; White, T. A.; Ulery, B. D., Instructive Design of Tri-Block Peptide Amphiphiles for Structurally Complex Micelle Formation. ACS Biomaterials Science & Engineering 2018, 30. Irvine, D. J.; Swartz, M. A.; Szeto, G. L., Engineering synthetic vaccines using cues from natural immunity. Nature materials 2013, 12 (11), 978-990. 31. Bachmann, M. F.; Jennings, G. T., Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns. Nature Reviews Immunology 2010, 10 (11), 787-796. 32. Missirlis, D.; Krogstad, D. V.; Tirrell, M., Internalization of p5314− 29 Peptide Amphiphiles and Subsequent Endosomal Disruption Results in SJSA-1 Cell Death. Molecular pharmaceutics 2010, 7 (6), 2173-2184. 33. Wang, T.-y.; Leventis, R.; Silvius, J. R., Artificially lipid-anchored proteins can elicit clustering-induced intracellular signaling events in Jurkat T-lymphocytes independent of lipid raft association. Journal of Biological Chemistry 2005, 280 (24), 22839-22846. 34. Kato, K.; Itoh, C.; Yasukouchi, T.; Nagamune, T., Rapid protein anchoring into the membranes of mammalian cells using oleyl chain and poly (ethylene glycol) derivatives. Biotechnology progress 2004, 20 (3), 897-904. 35. Liu, H.; Zhu, Z.; Kang, H.; Wu, Y.; Sefan, K.; Tan, W., DNA‐based micelles: synthesis, micellar properties and size‐dependent cell permeability. Chemistry–A European Journal 2010, 16 (12), 3791-3797. 36. Swee, L. K.; Guimaraes, C. P.; Sehrawat, S.; Spooner, E.; Barrasa, M. I.; Ploegh, H. L., Sortase-mediated modification of αDEC205 affords optimization of antigen presentation and immunization against a set of viral epitopes. Proceedings of the National Academy of Sciences 2013, 110 (4), 1428-1433. 37. Hirosue, S.; Kourtis, I. C.; van der Vlies, A. J.; Hubbell, J. A.; Swartz, M. A., Antigen delivery to dendritic cells by poly (propylene sulfide) nanoparticles with disulfide 26 ACS Paragon Plus Environment

Page 26 of 29

Page 27 of 29 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


38.

39.

40. 41.

42.

43. 44.

45.

46.

47. 48.

49.

50. 51.

conjugated peptides: Cross-presentation and T cell activation. Vaccine 2010, 28 (50), 78977906. Denton, A. E.; Wesselingh, R.; Gras, S.; Guillonneau, C.; Olson, M. R.; Mintern, J. D.; Zeng, W.; Jackson, D. C.; Rossjohn, J.; Hodgkin, P. D., Affinity thresholds for naive CD8+ CTL activation by peptides and engineered influenza A viruses. The Journal of Immunology 2011, 187 (11), 5733-5744. Pouniotis, D. S.; Esparon, S.; Apostolopoulos, V.; Pietersz, G. A., Whole protein and defined CD8+ and CD4+ peptides linked to penetratin targets both MHC class I and II antigen presentation pathways. Immunology and cell biology 2011, 89 (8), 904. Rudra, J. S.; Tian, Y. F.; Jung, J. P.; Collier, J. H., A self-assembling peptide acting as an immune adjuvant. Proceedings of the National Academy of Sciences 2010, 107 (2), 622-627. Fromen, C. A.; Rahhal, T. B.; Robbins, G. R.; Kai, M. P.; Shen, T. W.; Luft, J. C.; DeSimone, J. M., Nanoparticle surface charge impacts distribution, uptake and lymph node trafficking by pulmonary antigen-presenting cells. Nanomedicine: Nanotechnology, Biology and Medicine 2016, 12 (3), 677-687. Hamborg, M.; Rose, F.; Jorgensen, L.; Bjorklund, K.; Pedersen, H. B.; Christensen, D.; Foged, C., Elucidating the mechanisms of protein antigen adsorption to the CAF/NAF liposomal vaccine adjuvant systems: effect of charge, fluidity and antigen-to-lipid ratio. Biochimica et Biophysica Acta (BBA)-Biomembranes 2014, 1838 (8), 2001-2010. Yue, H.; Ma, G., Polymeric micro/nanoparticles: Particle design and potential vaccine delivery applications. Vaccine 2015, 33 (44), 5927-5936. Chiu, Y.-C.; Gammon, J. M.; Andorko, J. I.; Tostanoski, L. H.; Jewell, C. M., Modular vaccine design using carrier-free capsules assembled from polyionic immune signals. ACS biomaterials science & engineering 2015, 1 (12), 1200-1205. Xia, T.; Kovochich, M.; Liong, M.; Zink, J. I.; Nel, A. E., Cationic polystyrene nanosphere toxicity depends on cell-specific endocytic and mitochondrial injury pathways. ACS nano 2007, 2 (1), 85-96. Goodman, C. M.; McCusker, C. D.; Yilmaz, T.; Rotello, V. M., Toxicity of gold nanoparticles functionalized with cationic and anionic side chains. Bioconjugate chemistry 2004, 15 (4), 897-900. Wen, Y.; Waltman, A.; Han, H.; Collier, J. H., Switching the Immunogenicity of Peptide Assemblies Using Surface Properties. ACS nano 2016, 10 (10), 9274-9286. Fromen, C. A.; Robbins, G. R.; Shen, T. W.; Kai, M. P.; Ting, J. P.; DeSimone, J. M., Controlled analysis of nanoparticle charge on mucosal and systemic antibody responses following pulmonary immunization. Proceedings of the National Academy of Sciences 2015, 112 (2), 488-493. Zeng, Q.; Li, H.; Jiang, H.; Yu, J.; Wang, Y.; Ke, H.; Gong, T.; Zhang, Z.; Sun, X., Tailoring polymeric hybrid micelles with lymph node targeting ability to improve the potency of cancer vaccines. Biomaterials 2017, 122, 105-113. Jiang, H.; Wang, Q.; Sun, X., Lymph node targeting strategies to improve vaccination efficacy. Journal of Controlled Release 2017. Trevaskis, N. L.; Kaminskas, L. M.; Porter, C. J., From sewer to saviour [mdash] targeting the lymphatic system to promote drug exposure and activity. Nature Reviews Drug Discovery 2015, 14 (11), 781-803.



52. Liu, H.; Moynihan, K. D.; Zheng, Y.; Szeto, G. L.; Li, A. V.; Huang, B.; Van Egeren, D. S.; Park, C.; Irvine, D. J., Structure-based programming of lymph-node targeting in molecular vaccines. Nature 2014, 507 (7493), 519-522. 53. Yu, C.; An, M.; Li, M.; Liu, H., Immunostimulatory Properties of Lipid Modified CpG Oligonucleotides. Molecular pharmaceutics 2017, 14 (8), 2815-2823. 54. Jiang, H.; Wang, Q.; Sun, X., Lymph node targeting strategies to improve vaccination efficacy. Journal of Controlled Release 2017, 267, 47-56. 55. Joshi, V. B.; Geary, S. M.; Salem, A. K., Biodegradable particles as vaccine delivery systems: size matters. The AAPS journal 2013, 15 (1), 85-94. 56. Bachmann, M. F.; Jennings, G. T., Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns. Nature reviews. Immunology 2010, 10 (11), 787. 57. Reddy, S. T.; Van Der Vlies, A. J.; Simeoni, E.; Angeli, V.; Randolph, G. J.; O'Neil, C. P.; Lee, L. K.; Swartz, M. A.; Hubbell, J. A., Exploiting lymphatic transport and complement activation in nanoparticle vaccines. Nature biotechnology 2007, 25 (10), 1159-1164. 58. Ma, Z.; Li, J.; He, F.; Wilson, A.; Pitt, B.; Li, S., Cationic lipids enhance siRNA-mediated interferon response in mice. Biochemical and biophysical research communications 2005, 330 (3), 755-759. 59. Lindgren, M.; Hällbrink, M.; Prochiantz, A.; Langel, Ü., Cell-penetrating peptides. Trends in pharmacological sciences 2000, 21 (3), 99-103. 60. Wang, L.; Prakash, R. K.; Stein, C.; Koehn, R. K.; Ruffner, D. E., Progress in the delivery of therapeutic oligonucleotides: organ/cellular distribution and targeted delivery of oligonucleotides in vivo. Antisense and Nucleic Acid Drug Development 2003, 13 (3), 169189. 61. Yasun, E.; Li, C.; Barut, I.; Janvier, D.; Qiu, L.; Cui, C.; Tan, W., BSA modification to reduce CTAB induced nonspecificity and cytotoxicity of aptamer-conjugated gold nanorods. Nanoscale 2015, 7 (22), 10240-10248. 62. Almofti, M. R.; Harashima, H.; Shinohara, Y.; Almofti, A.; Baba, Y.; Kiwada, H., Cationic liposome-mediated gene delivery: biophysical study and mechanism of internalization. Archives of biochemistry and biophysics 2003, 410 (2), 246-253. 63. Mammadov, R.; Mammadov, B.; Toksoz, S.; Aydin, B.; Yagci, R.; Tekinay, A. B.; Guler, M. O., Heparin mimetic peptide nanofibers promote angiogenesis. Biomacromolecules 2011, 12 (10), 3508-3519. 64. Angeloni, N. L.; Bond, C. W.; Tang, Y.; Harrington, D. A.; Zhang, S.; Stupp, S. I.; McKenna, K. E.; Podlasek, C. A., Regeneration of the cavernous nerve by Sonic hedgehog using aligned peptide amphiphile nanofibers. Biomaterials 2011, 32 (4), 1091-1101. 65. Matson, J. B.; Stupp, S. I., Self-assembling peptide scaffolds for regenerative medicine. Chemical communications 2012, 48 (1), 26-33. 66. Ryan, D. M.; Nilsson, B. L., Self-assembled amino acids and dipeptides as noncovalent hydrogels for tissue engineering. Polymer Chemistry 2012, 3 (1), 18-33. 67. Webber, M. J.; Matson, J. B.; Tamboli, V. K.; Stupp, S. I., Controlled release of dexamethasone from peptide nanofiber gels to modulate inflammatory response. Biomaterials 2012, 33 (28), 6823-6832. 68. McDonald, K. L.; Webb, R. I., Freeze substitution in 3 hours or less. Journal of microscopy 2011, 243 (3), 227-233.


Page 28 of 29

Page 29 of 29 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


For Table of Contents use only Title: Peptide Amphiphile Micelle Vaccine Size and Charge Influence the Host Antibody Response Author: Rui Zhang, Josiah D. Smith, Brittany N. Allen, Jake S. Kramer, Martin Schauflinger, and Bret D. Ulery


Peptide Amphiphile Micelle Vaccine Size and Charge Influence the

Recommend Documents