Subscriber access provided by UNIV OF CONNECTICUT
Article
Ex Vivo Programming of Dendritic Cell by Mitochondria-Targeted Nanoparticles to Produce Interferon-Gamma for Cancer Immunotherapy Sean Marrache, Smanla Tundup, Donald A. Harn, and Shanta Dhar ACS Nano, Just Accepted Manuscript • DOI: 10.1021/nn403158n • Publication Date (Web): 30 Jul 2013 Downloaded from http://pubs.acs.org on August 5, 2013
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 Nano is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 31
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
Ex Vivo Programming of Dendritic Cell by Mitochondria-Targeted Nanoparticles to Produce Interferon-Gamma for Cancer Immunotherapy Sean Marrache,a§ Smanla Tundup,c§ Donald A. Harn,c and Shanta Dhara,b∗ a
NanoTherapeutics Research Laboratory, Department of Chemistry, bDepartment of Physiology and Pharmacology, University of Georgia, Athens, GA 30602, cDepartment of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602
∗Address correspondence to
[email protected] Keywords. Vaccine, photodynamic therapy, dendritic cell therapy, biodegradable polymer
ABSTRACT
One of the limitations for clinical applications of dendritic cell (DC)-based cancer immunotherapy is the low potency in generating tumor antigen specific T cell responses. We examined immunotherapeutic potential of a mitochondria-targeted nanoparticle (NP) based on a biodegradable polymer and zinc phthalocyanine (ZnPc) photosensitizer (T-ZnPc-NPs). Here, we report for the first time, that tumor antigens generated from treatment of breast cancer cells with T-ZnPc-NPs upon light stimulation activates DCs to produce high levels of interferon-gamma,
ACS Paragon Plus Environment
1
ACS Nano
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 31
an important cytokine considered as a product of T and natural killer cells. The remarkable ex vivo DC stimulation ability of this tumor cell supernatant is a result of interleukin (IL)-12/IL-18 autocrine effect. These findings contribute to the understanding of how in situ light activation amplifies the host immune responses when NPs deliver the photosensitizer to the mitochondria and open up the possibility of using mitochondria-targeted-NP treated, light activated cancer cell supernatants as possible vaccines.
ACS Paragon Plus Environment
2
Page 3 of 31
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
Host’s dysfunctional immune system assists tumors to evade immunosurveillance.1 However, the immune system when properly stimulated can eradicate ofcancer cells.2 Tumor immunity involves interactions of cytokines and effector cells. One cytokine that plays a central role in coordinating tumor immune responses is interferon-gamma (IFN-γ). Immature dendritic cells (iDCs) secrete cytokines to initiate and enhance both innate and acquired immunity, detect and capture antigens, undergo a maturation process, and prime immune responses by activating naïve T cells. Use of DCs for clinical cancer immunotherapy is extremely attractive.3,
4
DCs are
professional antigen presenting cells (APCs), which possess the entire array of antigenpresenting and co-stimulatory molecules. One of the major limitations for clinical application of DCs is the low potency in generating tumor antigen specific CD8+ T cell responses in vivo.5, 6 Infectious agents activate DCs to secret interleukin (IL)-12, which subsequently induces IFN-γ production by natural killer (NK) cells and directs T helper cell type 1 (Th1) development. We hypothesized that ex vivo engineering of DCs to produce IFN-γ for increased T-cell activity and development of a Th1-type immune response can serve as an alternative therapeutic modality for metastatic cancer. Photodynamic therapy (PDT), an anti-tumor therapeutic modality that has approval for the treatment of oncological diseases, involves administration of a photosensitizer (PS) followed by illumination of the tumor with a long wavelength (600-800 nm) light. PDT produces reactive oxygen species (ROS) resulting vascular shutdown, cancer cell apoptosis, and the induction of a host immune response.7, 8 Preclinical and clinical techniques of this method of cancer treatment are still being optimized to address that PDT sometimes fails to eradicate the targeted tumor and to boost immunity. We speculated that these failures may in part arise from inhomogeneous delivery of PS within the tumor, in particular the inability to reach its target organelle, the mitochondria of cells, and the inability to produce short-lived singlet oxygen (1O2)
ACS Paragon Plus Environment
3
ACS Nano
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 4 of 31
in the mitochondria of tumor cells. Those PS that accumulate inside the mitochondria induce apoptosis upon photo-irradiation, whereas those that bind to the non-mitochondrial compartments kill cells less efficiently by a non-apoptotic mechanism. Photodynamically induced mitochondrial apoptosis induces signal transduction pathways, which participate in the development of immune responses. Thus, we conjectured that exposing DCs ex vivo to tumor cell supernatant generated from mitochondria-targeted light activated cancer cells may unravel pathways which can be used to improve DC-based cancer immunotherapy. Thus we ask whether targeting mitochondria with an engineered biodegradable nanoparticle (NP) containing a mitochondria acting PS such as zinc phthalocyanine (ZnPc),9 induction of mitochondrial apoptosis after light stimulation, and activation of DCs ex vivo with cancer cell antigens can induce immunity. Polymeric NPs of poly(lactide-co-glycolide)-b-polyethyleneglycol (PLGA-bPEG) block copolymers are especially promising as drug delivery vehicles.10-16 We recently constructed a targeted functionalized polymer using PLGA-b-PEG and a lipophilic triphenyl phosphonium (TPP) cation17 for mitochondria-targeted delivery of payloads.18 Targeted-NPs from PLGA-b-PEG-TPP takes advantage of substantial negative mitochondrial inner membrane potential (Δψm) to deliver cargos into mitochondria.18 By using such an engineered vehicle to target the intracellular organelle mitochondria, we observed that DCs cultured with supernatants from breast cancer cells activated with mitochondria-targeted PDT had significant levels of IFN-
γ. To the best of our knowledge this is the first report, which demonstrates the ability of mitochondria-targeted system to stimulate murine bone marrow derived DCs (BMDCs) ex vivo to secret IFN-γ , the most versatile player of immune activation. By highlighting the importance of mitochondria targeted PDT of breast cancer and subsequent cytokine secretion by BMDCs receiving stimulation with mitochondria targeted PDT treated cancer cell, our data provide a
ACS Paragon Plus Environment
4
Page 5 of 31
model considering ex vivo manipulation of DC function for clinical applications. In this report, we discuss ex vivo IFN-γ production pathways from DCs upon stimulation with mitochondriatargeted-PDT-activated breast tumor cell antigens (Figure 1a). RESULTS AND DISCUSSION (b)
(a)
x
O O
O
y
O
PPh 3
O
n
PLGA-b-PEG-TPP Necrosis
Apoptosis
660 nm LASER
O
O
+ Engulf of apoptotic cells by immature DCs
PS* O2 ROS
N
N
N
N Zn
N
N
N
N
ZnPc
DC activation and maturation by a combination of apoptotic and necrotic cells
Immature DC
CD80, 86
MHC
Tumor antigen
T cell activation
Mature DC
(c) Diameter (nm) Zeta Potential (mV) Loading (%)
75 50 25 0
50
12
-25
10
40
8
30
6
20
4 10
2 0 0
0 10 20 30 40 50
ZnPc Weight (%)
10
20
30
40
50
0
T-ZnPc-NP
Encapsulation Efficiency (%)
ZnPc Weight (%)
(d)
Empty-T-NPs
500 nm
T-ZnPc-NPs
500 nm
T-ZnPc-NPs Diamater (nm) Zeta Potential (mV) Loading (%)
75 50 25 0
12
50
10
40
8
30
6 20
4
10
2
-25 0 10 20 30 40 50
0 0
10
ZnPc Weight (%)
20
30
40
50
0
Encapsulation Efficiency (%)
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 Nano
ZnPc Weight (%)
Empty-NT-NPs
500 nm
NT-ZnPc-NPs
500 nm
NT-ZnPc-NPs
Figure 1. (a) Action of mitochondria-targeted NPs upon light activation inside the mitochondria to produce ROS which causes cell death via apoptosis and necrosis, (b) Synthesis of T-ZnPc-NPs by following a nanoprecipitation method, (c) Size, zeta potential, and ZnPc loading in targeted and non-targeted NPs, and (d) TEM images of targeted and non-targeted empty and ZnPc-loaded NPs. ACS Paragon Plus Environment
5
ACS Nano
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 6 of 31
Construction of Mitochondria-Targeted NP for DC Activation. The use of mitochondriatargeted NPs for ZnPc delivery is obvious due to the fact that ZnPc acts on the mitochondria of cells and upon photo-activation generates short-lived 1O2 as the cytotoxic agent to show its photodynamic activity.19, 20 The second-generation ZnPc photosensitizer has strong photo-toxicity in the therapeutic window of 600-800 nm; however, the hydrophobic characteristic hinders its systemic administration and restricts its possible translation to the clinic. The active species involved in the ZnPc-based PDT, 1O2 has a short lifetime of
