Ovarian cancer immunotherapy using PD-L1 siRNA targeted delivery from folic acid-functionalized polyethylenimine: strategies to enhance T cell killing.

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Ovarian Cancer Immunotherapy Using PD-L1 siRNA Targeted Delivery from Folic Acid-Functionalized Polyethylenimine: Strategies to Enhance T Cell Killing Pei Yun Teo, Chuan Yang, Lynsey M. Whilding, Ana C. Parente-Pereira, John Maher, Andrew J. T. George, James L. Hedrick, Yi Yan Yang,* and Sadaf Ghaem-Maghami* is their ability to recognize cell surface antigens independently of human leukocyte antigen (HLA) compatibility.[2–5] However optimal T cell activity is dampened by various immunosuppressive mechanisms exerted by EOC cells as well as host immune cells in the cancer microenvironment.[6–8] In this regard, various studies have shown that interaction between programmed death-1 (PD-1) and its ligand PD-L1 is responsible for T cell hyporesponsiveness in EOC.[6–9] PD-L1 protein is over expressed on ovarian cancer cells, especially those of high-grade serous histology, as well as tumour associated monocytes.[8–10] High grade serous ovarian cancer is the most common and aggressive subtype and accounts for almost 70% of ovarian cancer deaths.[1] PD-L1 is also expressed on healthy tissues, activated dendritic cells, T cells, B cells, macrophages, natural killer cells, mesenchymal stem cells, cultured bone marrowderived mast cells, activated vascular endothelial cells,[11]and immune privileged sites including the placenta and the eyes, where it plays a role in preventing autoimmunity.[9,11–13] Earlier work from Maine et al. showed that PD-L1 expression on EOC cells as well as patient derived ascitic monocytes interacted with PD-1 expressed on activated T cells and dampened their function, suggesting the role of PD-L1/PD-1 pathway as an immune-escape route for EOC.[8] In addition, Hamanishi et al. demonstrated that PD-L1 expressed by EOC tissue not only inhibited CD8+ tumour infiltrating lymphocytes (TILs),

Adoptive T cell immunotherapy is a promising treatment strategy for epithelial ovarian cancer (EOC). However, programmed death ligand-1 (PD-L1), highly expressed on EOC cells, interacts with programmed death-1 (PD-1), expressed on T cells, causing immunosuppression. This study aims to block PD-1/PD-L1 interactions by delivering PD-L1 siRNA, using various folic acid (FA)–functionalized polyethylenimine (PEI) polymers, to SKOV-3-Luc EOC cells, and investigate the sensitization of the EOC cells to T cell killing. To enhance siRNA uptake into EOC cells, which over express folate receptors, PEI is modified with FA or PEG–FA so that siRNA is complexed into nanoparticles with folate molecules on the surface. PEI modification with a single functional group lowers the polymer cytotoxicity compared to unmodified PEI. FA-conjugated polymers increase siRNA uptake into SKOV-3-luc cells and decrease unspecific uptake into monocytes. All polymers result in 40% to 50% PD-L1 protein knockdown. Importantly, SKOV-3-Luc cells treated with either PEI–FA or PEI– polyethylene glycol (PEG)–FA/PD-L1 siRNA complexes are up to twofold more sensitive to T cell killing compared to scrambled siRNA treated controls. These findings are the first to demonstrate that PD-L1 knockdown in EOC cells, via siRNA/FA-targeted delivery, are able to sensitize cancer cells to T cell killing.

1. Introduction Epithelial ovarian cancer (EOC) remains the most lethal gynaecological cancer despite advances in radical surgery and combination chemotherapy. The poor prognosis of ovarian cancer is mainly attributed to the eventual development of chemo resistance.[1] To meet this clinical need, adoptive cell therapy, involving the transfer of T cells, transduced with chimeric antigen receptors (CAR T cells), has been investigated as an alternative treatment for EOC. An advantage of CAR T cells

P. Y. Teo, Dr. C. Yang, Dr. Y. Y. Yang Institute of Bioengineering and Nanotechnology 31 Biopolis Way, The Nanos, Singapore 138669, Singapore E-mail: [email protected] P. Y. Teo, Dr. L. M. Whilding, Dr. S. Ghaem-Maghami Imperial College London Hammersmith Campus Du Cane Road, London, W12 0NN, UK E-mail: [email protected]

DOI: 10.1002/adhm.201500089

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Dr. L. M. Whilding, Dr. A. C. Parente-Pereira, Dr. J. Maher King’s College London Guy’s Hospital St Thomas Street, London SE1 9RT, UK Prof. A. J. T. George Brunel University Kingston Lane Uxbridge, Middlesex UB8 3PH, UK Dr. J. L. Hedrick IBM Almaden Research Center 650 Harry Road, San Jose, CA 95120, USA

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Adv. Healthcare Mater. 2015, 4, 1180–1189

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scrambled siRNA (as a negative control) or PD-L1 siRNA was compared after co-culture with a previously developed CAR approach, named T4 immunotherapy.[5] In T4 immunotherapy, T-cells are engineered to co-express a CAR (T1E28z) that targets the extended ErbB family. T4 engineered T-cells have shown potent cytotoxic effects against EOC cells, which have upregulated expressions of ErbB 1, 2, and 3.[5] The effects of PD-L1 down regulation of SKOV-3-Luc on T4 functionality was also studied by measuring interferon-γ (IFN-γ) production by T4 cells and expression of the degranulation marker, CD107a on T4 cells. To our knowledge, this work is the first to demonstrate PD-L1 siRNA/FA-targeted delivery into EOC cells for immunosensitization to T cell killing.

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it also inversely correlated with patient survival.[9] Monoclonal antibodies, aimed at blocking PD-1/PD-L1 interaction, have shown tremendous potential to reverse T cell hyporesponsiveness.[10,13–17] The blockade of the PD-1/PD-L1 immune checkpoint pathway is therefore an attractive approach to enhance T cell mediated tumour regression in EOC. While extensive work focused on PD-1/PD-L1 blocking monoclonal antibody therapies, few studies have explored the use of genetic interventions such as PD-L1 targeted siRNA. Successful stimulation of the immune system using anti PD-1 or PD-L1 antibodies suggests that PD-L1 knockdown in EOC cells may also sensitize the cancer to T cell killing. Synthetic polymers, such as polyethylenimine (PEI),[18] cyclodextrin-based polycations,[19] and polyphosphates[20] have been used for siRNA delivery due to low costs, designability and ease of production.[21] Among the polymers, PEI, together with its derivatives, have shown high efficacy of siRNA delivery both in vitro and in vivo.[22,23] The success of PEI is attributed mainly to its high cationic charge as well as its composition of primary, secondary and tertiary amines. The high charge density of PEI enables it to bind and compact siRNA into nanocomplexes, protecting siRNA from enzymatic degradation.[24] Tertiary amines on PEI are able to sequester protons in the endosomes, enabling PEI/siRNA complexes to escape from lysosomes in a process known as the “proton sponge hypotheses.”[25] The abundance of amine groups also allows conjugation of functional moieties to tune polymer properties such as cytotoxicity, targeting ability, and specificity, making PEI a very versatile gene carrier.[23,26,27] However, a major drawback of PEI as a gene carrier is its cytotoxicity caused by its high cationic charge density which depolarizes mitochondrial membranes.[28] Since PD-L1 is also expressed on healthy tissues/cells,[9,11–13] it is important to deliver PD-L1 targeted siRNA specifically to EOC cells. To enhance siRNA uptake into EOC cells, which over express folic acid (FA) receptors,[29,30] PEI was modified with FA and PEG–FA and used to complex with siRNA into nanoparticles with folate molecules on the surface. FA was designed to enhance cellular uptake of siRNA through receptor-mediated endocytosis,[31] whereas PEG was used to provide stability in a serum-containing medium.[32] It is envisioned that the polymer/ siRNA nanocomplexes can be delivered to EOC based on the enhanced permeability and retention effect at tumor tissues[33] and specifically taken up by the EOC cells via folate receptormediated endocytosis. A disulfide bond (-SS-) was built between PEG–FA and PEI to render glutathione-sensitive cleavage of PEG–FA or PEG from the polymer/siRNA nanocomplexes inside the cells which has been shown to enhance gene transfection.[34,35] The polymer/siRNA nanocomplexes were used to knockdown PD-L1 on a luciferase expressing SKOV-3 (SKOV3-Luc) EOC cell line, and to determine the knockdown effects on the anti-tumor efficacy and functionality of CAR T cells. In addition, the specificity of particle uptake into SKOV-3-Luc cells was investigated when challenged with the presence of human primary peripheral blood mononuclear cells (PBMCs). The PBMCs consists of professional phagocytes, such as monocytes, which typically eliminate nanoparticles preventing them from reaching target cells.[36] It is therefore important to design nanoparticles which evade the unspecific uptake by monocytes in the bloodstream. The viability of SKOV-3-Luc cells treated by

2. Results 2.1. Modification and Characterization of PEI To study the effects of FA and PEG conjugation on PEI for siRNA delivery, methylcarboxytrimethylene carbonate (MTC)– SSPEG and MTC–SSPEG–FA were designed and synthesized (Figure S1, Supporting Information). PEI was modified with these MTC monomers in a 1:1 molar ratio to synthesize PEI–PEG and PEI–PEG–FA, respectively (Scheme 1A,B). The reaction is fast, highly controllable, and correlated well to feed molar ratios of PEI to MTC monomers as verified by 1H NMR analysis. For example, the conjugation degree of MTC–PEG– FA modification of PEI was found to be 1:1.1 by comparing the relative integral intensities of benzyl signals (7.5–7.7 ppm) and ether signals (3.6 ppm) of MTC–PEG–FA with PEI methylene signals (2.4–2.9 ppm) (Scheme 1B). In addition, PEI–FA, where FA was directly conjugated to the PEI backbone, was synthesized using 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/sulfo-N-hydroxysuccinimide (NHS) chemistry. 1H NMR analysis showed that PEI–FA composition of 1:1 corresponded well with feed molar ratios (Scheme 1C). The chemical compositions of modified PEI are summarized in Table 1. The synthesized polymers were characterized for their binding ability with siRNA, particle size and surface charge. The siRNA binding ability of modified and unmodified PEI polymers was investigated by the SYBR green siRNA binding assay. SYBR green intercalates with unbound siRNA and emits a fluorescence which can be measured by a plate reader. Polymer/siRNA binding would be indicated by a lowered fluorescence. Figure S4, Supporting Information, shows that the minimum N/P ratio needed to maximally reduce fluorescence is N/P ratio 8 for polymers without PEG (PEI and PEI–FA) and 10 for polymers with PEG (PEI–PEG and PEI–PEG–FA). Therefore polymer/siRNA complexes formed at N/P ratios of 10 and above were tested for PD-L1 knockdown efficiencies in SKOV3-Luc cells. To measure size and zeta potential, polymer/siRNA complexes were formed and monitored by dynamic light scattering (DLS) measurements. PEI with PEG formed larger sized particles (167–244 nm) with siRNA than PEI without PEG (104– 110 nm) (Table 1). In addition, the zeta potential measurements show that modified PEI/siRNA complexes had a reduction in



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Scheme 1. Synthesis of modified PEI and chemical compositions. PEI reacted with MTC–PEG or MTC–PEG–FA or FA in 1:1 molar ratio to form A) PEI–PEG, B) PEI–PEG–FA, or C) PEI–FA, respectively. 1H NMR spectra of PEI–PEG–FA and PEI–FA in D2O.

positive charge, but still remained cationic (22–29 mV), compared to unmodified PEI/siRNA particles (≈37 mV) (Table 1).

2.2. Cytotoxicity of PEI/siRNA Complexes The cytotoxicity of the polymer/siRNA complexes was investigated by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) cell viability assay. The cell viability of SKOV-3-Luc cells was lower when treated with unmodified PEI/ siRNA complexes compared to modified PEI/siRNA complexes at N/P 40 and above (Figure 1). The modification of PEI therefore allowed higher N/P ratios to be used to maximize PD-L1 knockdown while maintaining high cell viability. Modified PEI polymers, with lowered toxicity, are thus better siRNA transfecting agents than their unmodified counterpart.

2.3. PD-L1 Knockdown The in vitro PD-L1 knockdown study was performed by treating SKOV-3-Luc cells with polymer/siRNA complexes formed at N/P ratios ranging from 10 to 80 and then analysing the PD-L1 cell surface expression by flow cytometry. In general, it is observed that the higher the N/P ratio, the greater the PD-L1 knockdown efficiency (Figure 2A). While all polymers mediated PD-L1 knockdown, polymers without PEG (PEI and PEI–FA) had higher transfection

efficiency and resulted in a lower PD-L1 protein expression (51%–55%) on SKOV-3-Luc cells compared to 62%–65% for the polymers with PEG (PEI–PEG and PEI–PEG–FA), at N/P ratios of 50 and 80 (Figure 2A). Confocal images showed that the expression of PD-L1 on the surface of SKOV-3-Luc cells was reduced after treatment with PEI–FA/PD-L1 siRNA complexes compared to treatment with PEI–FA/scrambled siRNA complexes (Figure 2E). Western blot data also demonstrated PD-L1 knockdown in SKOV-3-Luc cells after treatment with PEI–FA/ PD-L1 siRNA complexes as compared to untreated and PEI– FA/scrambled siRNA treated controls (Figure 2F). To determine transfection efficiency, PD-L1 mRNA levels were determined by real-time quantitative polymerase chain reaction (qRT-PCR). Polymer/siRNA complexes resulted in final PD-L1 mRNA expressions of 17.4%–40%, with PEI–FA and PEI–PEG–FA polymers mediating the greatest knockdowns (Figure 2B). The expression levels of mRNA (17.4%–40%) and protein (53%–65%) varied considerably (Figure 2A,B). This observation, which has also been reported by other groups,[37,38] is not surprising as protein knockdown and expression are not solely dependent on mRNA degradation but also on processes of translation and protein degradation. To study the duration of PD-L1 protein knockdown, expression of PD-L1 on SKOV-3-Luc cells was analysed for up to 6 days post transfection using PEI–FA/siRNA complexes. The time point study correlates to the experimental duration of SKOV-3-Luc/T cell co-culture experiments. Figure 2C,D shows that a single treatment with PEI–FA/siRNA complexes on

Table 1. Physical properties of modified and unmodified PEI/siRNA complexes. Mole ratioa)

Polymer ID

PDIb)

Zetab) [mV]

PEI

…

110 ± 4

0.19

36.8 ± 2.3

PEI–FA

1:1

104 ± 2

0.29

28.6 ± 1.6

PEI–PEG

1:0.8

244 ± 2

0.35

23.1 ± 2.6

PEI–PEG–FA

1:1.1

167 ± 2

0.26

22.8 ± 1.2

a)PEI:modified

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Sizeb) [nm]

group mole ratio based on 1 mol PEI = 10 000 g; b)Size, PDI, and zeta potential measurements are of polymer/siRNA complexes formed at N/P ratio 50.





Cell viability (%)

PEI-FA

PEI-PEG *

PEI-PEG-FA **

**

80 60 40 20 0 10

20

30

40

50

80

N/P ratio

Figure 1. Viability of SKOV-3-Luc cells after treatment with modified or unmodified PEI/siRNA complexes at various N/P ratios. Results represent mean ± SEM of three independent experiments. Student’s T test was used to calculate significance, (*p < 0.05, **p < 0.01).

SKOV-3-Luc cells was sufficient to maintain low PD-L1 protein expression (≈60% protein expression) as well as mRNA expression (≈20%) for up to 6 days post transfection.

2.4. Selective Uptake To investigate whether modification of PEI increased selective uptake of polymer/siRNA complexes into tumour cells, polymer/Alexa Fluor 488 labelled siRNA complexes were incubated with 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) labelled SKOV-3-Luc cells that were co-cultured with PBMCs in 1:1 ratio. The uptake of siRNA in cells was quantified using median fluorescent intensity (MFI) measured by flow cytometry. To study the uptake of siRNA in SKOV-3-Luc cells, MFI of Alexa Flour labelled siRNA was measured in the CFSE-positive cell population. SKOV-3-Luc cells incubated with modified PEI/siRNA complexes had higher MFI values (234 ± 4 to 245 ± 21) compared to cells incubated with unmodified PEI/siRNA complexes (186 ± 15) (Figure 3A). To investigate siRNA uptake in PBMCs, MFI of siRNA was determined in the CFSE-negative cell population. The majority of siRNA uptake was observed in the phagocytic monocytes, which had MFI values ranging from 402 ± 8 to 556 ± 52 (Figure 3B). The MFI of intracellular siRNA in lymphocytes however was low and did not vary with the use of different polymers (data not shown). The lymphocyte population was thus excluded from the analysis. Figure 3B shows that monocytes incubated with PEI–FA/siRNA or PEI–PEG–FA/siRNA complexes resulted in lower MFI values (402 ± 8 and 446 ± 45, respectively) compared to unmodified PEI/siRNA complexes (509 ± 36) (Figure 3B). It is unsurprising that the monocytes, which are professional phagocytic cells, showed a higher uptake of siRNA than SKOV-3-Luc tumor cells. Despite the phagocytic nature of the monocytes, PEI–FA and PEI–PEG–FA demonstrated lower unspecific uptake of siRNA into monocytes compared to PEI/siRNA and PEI–PEG/siRNA complexes. The incorporation of FA not only increased siRNA uptake into SKOV-3-Luc cells, it also decreased unspecific uptake in monocytes (Figure 3A,B).


2.5. Effects of PD-L1 siRNA Delivery on T4 Responses and Functionality To determine whether blocking PD-1/PD-L1 interactions by PD-L1 siRNA delivery to SKOV-3-Luc cells using either PEI–FA (Figure 4A) or PEI–PEG–FA (Figure 4B) polymers enhanced T4 cell killing, the viability of SKOV-3-Luc cells was analysed by the MTT assay. In addition, anti-PD-L1 blocking antibodies were added to PEI–FA or PEI–PEG–FA/siRNA complexes-treated cells to further decrease PD-1/PD-L1 interactions. Figure 4 shows that SKOV-3-Luc cells treated with either PEI–FA or PEI–PEG–FA/PD-L1 siRNA complexes and control antibodies were significantly more susceptible to T4 killing than SKOV-3-Luc cells treated with PEI–FA or PEI–PEG–FA/ scrambled siRNA complexes and control antibodies, respectively, at 48 and 72 h. The difference in viability of SKOV-3-Luc cells treated with PEI–FA and PEI–PEG–FA is due to biological variation of T cell killing efficiency from different donors and not due to differences in toxicity of polymer/siRNA treatment, which were shown to be similar in Figure 1. Moreover, pre-incubation of PD-L1 knockdown SKOV-3-Luc cells with anti-PD-L1 antibody, before starting SKOV-3-Luc/T4 co-culture, enhanced SKOV-3-Luc death in response to T4 presence at 48 and 72 h (Figure 4). To validate these findings, it is necessary to determine whether PD-L1 knockdown affects SKOV-3-Luc cell viability. Figure S5, Supporting Information, shows that viability of SKOV-3-Luc cells is independent of PD-L1 knockdown, verifying that the reduction of SKOV-3-Luc cell viability, demonstrated in Figure 4, was T4 cell mediated. In addition, Figure 4 shows that SKOV-3-Luc cell viability was lower when SKOV-3-Luc cells were treated with both PD-L1 siRNA and anti-PD-L1 antibody compared to when they were treated with scrambled siRNA and anti-PD-L1 antibody. The observation that PD-L1 siRNA treatment further decreased SKOV-3-Luc cell viability after pre-treatment with anti-PD-L1 antibody is unexpected as the concentration (50 µg mL−1) of anti-PD-L1 antibody used was at saturation levels (Figure S6, Supporting Information). Treatment with the antibody alone should theoretically have maximally blocked all PD-1/PD-L1 interactions leading to maximal T cell cytotoxicity. The additive cytotoxicity effect observed suggests that T4 cells might be activated via different pathways when PD-L1 siRNA and anti-PD-L1 antibodies were used. To elucidate how T4 cells mediate enhanced killing of SKOV-3-Luc cells with PD-L1 knockdown, two cytotoxic T cell functions were examined- production of gamma interferon (IFN-γ) and degranulation ability for the release of perforin and granzymes. IFN-γ and secreted granzymes trigger apoptosis by the induction of pro-apoptotic molecules such as surface expression of Fas and its ligands as well as activation of caspases.[39,40] The IFN-γ concentration in co-culture supernatant was determined by ELISA. Figure 5A shows that T4 cells, in co-culture with SKOV-3-Luc cells treated with PEI–FA/PD-L1 siRNA complexes and control antibody, secreted significantly more IFN-γ than when they were in co-culture with SKOV-3-Luc cells treated with PEI–FA/scrambled siRNA complexes and control antibody at 48 and 72 h. When anti-PD-L1 antibody was added to PD-L1 knockdown SKOV-3-Luc cells, the concentration of



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Figure 2. A,C) PD-L1 protein and B,D) mRNA expression of SKOV-3-Luc cells expressed as a percentage of polymer/scrambled siRNA complexestreated cells. A) PD-L1 protein exprerssion 3 days post transfection using polymer/siRNA complexes at various N/P ratios. B) PD-L1 mRNA expression at 3 days post transfection with polymers/siRNA complexes at N/P 50. C) PD-L1 protein and D) mRNA expression at various time points post transfection using PEI–FA/siRNA at N/P 50. Results represent mean ± SEM of four independent experiments. E) Confocal images of SKOV-3-Luc cell surface PD-L1 (red) expression after treatment with PEI–FA/PD-L1 siRNA compared to PEI–FA/scrambled siRNA treated control. Cell nuclei (blue) are stained with Hoechst 33342. Scale bar: 100 nm. F) Western blot images of PD-L1 protein levels in SKOV-3-Luc cells 3 days post transfection with PEI–FA/scrambled or PD-L1 siRNA complexes formed at N/P 50.

IFN-γ was further increased after 48 h but not after 72 h of coculture (Figure 5A). The degranulation ability of T4 cells was analyzed by the expression of lysosomal marker, CD107a, on T4 cell surface after co-culture with SKOV-3-Luc cells. T4 cells co-cultured with SKOV-3-Luc cells treated with PEI–FA/PD-L1 siRNA complexes and control antibody had significantly increased CD107a expression than when co-cultured with SKOV-3-Luc cells treated with PEI–FA/scrambled siRNA complexes and control antibody at 48 h (Figure 5B). The addition of anti-PD-L1 blocking antibodies to scrambled siRNA treated SKOV-3-Luc cells also caused T4 cells to express higher levels of CD107a but no additive effect was observed when SKOV-3-Luc cells were dual treated with both PD-L1 siRNA and anti-PD-L1 antibody (Figure 5B).

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3. Discussion PD-L1 is an attractive knockdown target as it has demonstrated tremendous therapeutic potential in various cancers and has resulted in mild immune-related adverse events (IRAE), characterized by surges in serum inflammatory cytokines and T cell inflammatory infiltration of solid organs, when PD-1/PD-L1 interactions were blocked.[14,15,17,41] Recently, Keytrumab (pembrolizumab) a PD-1 blocking antibody, received Food and Drug Administration (FDA) approval for treating advanced melanoma.[42] As PD-L1 is also expressed on healthy tissues, such as the placenta and eye, there is a need for targeted delivery of PD-L1 siRNA to cancer tissues. In this work, we have synthesized various FA and PEG conjugated PEI polymers for




600

* siRNA uptake into monocytes (MFI)

siRNA uptake into SKOV-3-Luc cells (MFI)

B

250 200 150 100 50

siR

*

500 400 300 200 100 0

0 NA

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A 300

a

e lon

I

PE

P

FA EI-

FA EG GI-P PE PE I PE

ne

NA

alo

I PE

siR

A

I-F

PE

G FA PE GE I-P PE

IPE

Figure 3. Intracellular uptake of Alexa Fluor 488 tagged siRNA into CFSE A) labelled SKOV-3-Luc cells or B) unlabeled monocytes. SKOV-3-Luc cells were co-cultured with PBMCs (includes monocytes) at 1:1 ratio and treated with modified or unmodified PEI/Alexa Fluor 488 tagged siRNA complexes, formed at N/P ratio 50. MFI of internalized siRNA in individual cell populations was measured using flow cytometry by gating on the CFSE positive (SKOV-3-Luc) or negative populations (monocyte). Results represent mean ± SEM of three individual experiments.

targeted PD-L1 siRNA delivery to EOC cells, which over express FA receptors.[29,30] We showed that modification of PEI resulted in polymers with lowered cytotoxicity, which mediated ≈40%–50% knockdown of PD-L1 expression. FA modified PEI (PEI–FA and PEI–PEG–FA) demonstrated higher specificity of uptake into tumour cells and this was especially enhanced when PEI–FA was used. Furthermore, we demonstrated the pre-clinical activity of PEI–FA or PEI–PEG–FA/PD-L1 siRNA complexes which rendered tumour cells more susceptible to T4 cell killing, compared to PEI–FA or PEI–PEG–FA/scrambled siRNA complexes. The increase of IFN-γ cytokine release and degranulation of perforin and granzymes by T4 cells have been attributed to cause enhanced SKOV-3-Luc cell killing. PEI was successfully modified with various functional groups (FA, PEG, PEG–FA) using either EDC/NHS conjugation or the functional cyclic carbonate monomers to form polymers with well-defined compositions (PEI–FA, PEI–PEG,

B

SKOV-3-Luc viability normalized to untreated control (%)

80

60

Scrambled siRNA + control Ab Scrambled siRNA + anti PD-L1 Ab PD-L1 siRNA + control Ab PD-L1 siRNA + anti PD-L1 Ab **

40

*

**

20

80 SKOV-3-Luc viability normalized to untreated control (%)

A

and PEI–PEG–FA). Characterization studies showed that all the polymers completely bound siRNA at N/P ratio 10, formed polymer/siRNA complexes with sizes below 200 nm (with the exception of PEI–PEG) and have cationic surface charges. In general, nanoparticles smaller than 200 nm are desirable due to enhanced permeability and retention (EPR) effect at tumor tissues,[33] and higher cellular uptake by clathrin-mediated endocytosis.[43] PEG conjugated PEI displayed larger sizes as the flexibility and folding of PEG chains have been postulated to reduce the compacting of the particle.[44] Cationic particles are beneficial for cellular uptake due to the electrostatic interaction with anionic proteoglycans, such as sialic acid and syndecan, which lead to endocytosis.[45] On the other hand, cationic charge can cause cytotoxicity. A single modification of PEI with FA, PEG or PEG–FA significantly reduced cytotoxicity of PEI (Figure 1). This may be due to the lowered charge density of modified PEI/siRNA complexes compared to unmodified PEI

60

Scrambled siRNA + control Ab Scrambled siRNA + anti PD-L1 Ab PD-L1 siRNA + control Ab PD-L1 siRNA + anti PD-L1 Ab * *

40

20

0

0 72 48 SKOV-3-Luc/ T4 (1:1) incubtaion time (h)

48

72

SKOV-3-Luc/T4 (1:1) incubation time (h)

Figure 4. Viability of SKOV-3-Luc cells after co-culture with T4 cells. SKOV-3-Luc cells were treated with A) PEI–FA/PD-L1 siRNA complexes or B) PEI– PEG–FA/PD-L1 siRNA complexes formed at N/P 50 and control or anti-PD-L1 antibody was added at 3 days post transfection. T4 cells were added in a 1:1 ratio to cancer cells and incubated for either 48 or 72 h. Results represent mean ± SEM of three independent experiments. Student’s T test was used to calculate significance, (*p < 0.05, **p < 0.01).




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A 1400 1200 IFN-gamma (pg/mL)

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1000

T4 alone Scramble siRNA + control Ab Scramble siRNA + anti PD-L1 Ab PD-L1 siRNA + control Ab PD-L1 siRNA + anti PD-L1 Ab

**

800 **

600 400

*

200 0 48 72 SKOV-3-Luc/T4 (1:1) incubation time (h)

B CD107a expression (MFI)

60 50 40

T4 alone Scramble siRNA + control Ab Scramble siRNA + anti PD-L1 Ab PD-L1 siRNA + control Ab PD-L1 siRNA + anti PD-L1 Ab * *

30 20 10 0

48 72 SKOV-3-Luc/T4(1:1) incubation time (h)

Figure 5. INF-γ release from T4 cells. A) and expression of degranulation marker CD107a on T4 cells. B) after co-culture with SKOV-3-Luc cells treated with PEI–FA/siRNA complexes and anti-PD-L1 and antibody. Results represent mean ± SEM of three independent experiments. Student’s T test was used to calculate significance, (*p < 0.05, **p < 0.01).

(Table 1). Functional group modification on the PEI has been reported to decrease the charge density of PEI due to the conversion of positively charged amines to amide groups which have a neutral charge.[27] Yang et al. and Thomas et al. reported similar reduction of PEI cytotoxicity after functional group modification.[27,46] For PD-L1 knockdown, polymers without PEG resulted in higher PD-L1 knockdown compared to PEGylated polymers (Figure 2A). This finding is consistent with previous work documenting that PEGylated polymers decreased nucleic acid transfection efficiencies.[47,48] Although both PEI and PEI–FA/ PD-L1 siRNA complexes resulted in similar levels PD-L1 knockdown, PEI–FA/siRNA complexes have a lower toxicity profile compared to unmodified PEI/siRNA complexes (Figure 1). In addition, PD-L1 levels maintained low for up to 6 days post transfection (Figure 2C,D). The maintenance of lowered PD-L1 expression levels compared to scrambled siRNA-treated controls ensured that any observed changes in SKOV-3-Luc cell viability and T4 function were due to differences in PD-L1 expression on SKOV-3-Luc cells in SKOV-3-Luc/T4 co-culture experiments. It was also demonstrated that PD-L1 knockdown on its own did not affect cell viability of SKOV-3-Luc cells (Figure S5, Supporting Information). Both FA conjugated polymers demonstrated increased siRNA uptake into SKOV-3-Luc cells and a reduction in unspecific uptake into monocytes. This observation was more pronounced when PEI–FA polymer was used. FA conjugation on nanoparticles has been reported to enhance cellular uptake.[49,50]

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For SKOV-3-Luc/T4 cell co-culture studies, both PEI–FA and PEI–PEG–FA polymers were used to deliver PD-L1 siRNA to SKOV-3-Luc cells. PD-L1 knockdown SKOV-3-Luc cells, regardless of polymer used, were more susceptible to T4 killing than scrambled siRNA treated SKOV-3-Luc cells (Figure 4). This effect was enhanced when PD-L1 knockdown SKOV-3-Luc cells were pre-incubated with anti-PD-L1 antibodies before coculture with T4 cells, emphasizing that the role PD-1/PD-L1 interactions play in controlling T4 responses (Figure 4). Interestingly, an additive effect of SKOV-3-Luc cell viability reduction was observed when SKOV-3-Luc cells, pre-treated with anti-PD-L1 antibody, were treated with PEI–FA/PD-L1 siRNA complexes. This indicates different mechanisms of siRNA and blocking antibody, which limit de novo production of PD-L1 and block existing PD-L1 interactions, respectively. Blocking PD-1/ PD-L1 interactions caused tumour cell death by the increased T4 production of IFN-γ and release of cytotoxic perforin and granzymes (Figure 5). Addition of anti-PD-L1 antibody to PD-L1 knockdown SKOV-3-Luc cells caused an increased production of IFN-γ at 48 h but not at 72 h (Figure 5A). This may be explained by lowered T4 cell antigen stimulation at 72 h time point due to tumour cell death (Figure 4), thus slowing T4 production of IFN-γ. No additive effect was observed for the expression of CD107a at both time points when SKOV3-Luc cells were treated with anti-PD-L1 antibody on top of PD-L1 knockdown (Figure 5B). This may be due to the limitations of the CD107a assay which is an indirect measure of degranulation and does not quantify concentrations of cytotoxic granzymes and perforin. These results are in accordance with other PD-1/PD-L1 blocking studies using virus specific CD8+ T cells in lymphocytic choriomeningitis virus (LCMV) mouse models, where treatment with anti-PD-L1 antibodies increased both IFN-γ and degranulation, leading to enhanced T cell responses against viral infection.[51,52] The results show that both PEI–FA and PEI–PEG–FA/PD-L1 siRNA complexes were able to effectively knockdown PD-L1 on EOC cells to increase CAR T cell functionality and killing of EOC cells compared to the respective PEI–FA and PEI–PEG–FA/scrambled siRNA treated controls. As PEG has been shown to improve serum stability, future studies would involve the investigation of how the number of PEG–FA chains conjugated on the PEI backbone would perform in vivo.[32]

4. Conclusion This study demonstrates that PEI–FA and PEI–PEG–FA polymers successfully deliver PD-L1 siRNA into EOC cells, which effectively blocks PD-1/PD-L1 interactions and thus enhances T cell immunotherapy for EOC treatment. The modification of PEI with FA or PEG–FA not only lowers cytotoxicity, but it also allows for tumour cell targeting towards EOC cells, highlighting their potential for use as gene delivery carriers.

5. Experimental Section Materials: Branched polyethylenimine (PEI, Mn 10 kDa, Sigma-Aldrich, Gillingham, U.K.) was freeze dried before use. PD-L1 and scrambled siRNA





(MES) buffer (3 mL) and stirred for 2 h at room temperature. PEI–FA was purified by ultracentrifugation (MWCO 10 kDa) and freeze dried. PEI–FA, 1H NMR (400 MHz, D2O, 22 °C): δ 8.50 (s, 1H, H-a), 7.56 (d, 2H, H-b), 6.67 (d, 2H, H-c), 2.27–3.93 (m, 930H, H of PEI) (Scheme 1C). Particle Size and Zeta Potential Measurements: PD-L1 siRNA was complexed with modified and unmodified PEI in RNAse free water at N/P 50, defined as the ratio between the nitrogen content of the polymer and the phosphorous content of the siRNA, by mixing siRNA solution with polymer solution. The mixture was incubated for 30 min at room temperature. Samples were diluted to 1 mL using RNAse free water. Size and zeta potential were analysed by dynamic light scattering (DLS) using the Zetasizer Nano (Malvern Instrument Ltd., Worcestershire, UK) equipped with He–Ne laser. Scattered light was detected at a set temperature of 25 °C and at an angle of 90°. Particle size and zeta potential measurements were repeated for 3 runs per sample and results indicate mean ± standard deviation of 3 readings. Cytotoxicity Studies of Polymer/siRNA Complexes: Cytotoxicity studies were performed using the thiazolyl blue tetrazolium bromide (MTT) assay. SKOV-3-Luc cells seeded in 96 well plates at 1.3 × 104 cells per well were treated with polymer/scrambled siRNA complexes formed as described earlier. 72 h post treatment, cells were incubated for 2 h at 37 °C with MTT solution (0.5 mg mL−1 in cell culture medium). Formazan crystals formed were dissolved in DMSO (100 µL) and absorbance was read using a microplate spectrophotometer (Molecular Devices, Wokingham, UK) at wavelengths of 550 and 690 nm. Relative cell viability was expressed as [(Absorbance550 – Absorbance690)sample/(Absorbance550 – Absorbance690)control] × 100%. Data are expressed as mean ± SEM of at least eight replicates per N/P ratio from three independent experiments. Flow Cytometry: SKOV-3-Luc cells were seeded in 24 well plates at 8 × 104 cells per well. Polymer/PD-L1 siRNA complexes were prepared as described above at various N/P ratios and added to cultured cells (final siRNA concentration: 200 × 10−9 M). Following 4 h of incubation, the medium was replaced and the cells were analysed for PD-L1 expression 3, 4, or 6 days post transfection by flow cytometry (FACSCalibur cytometer with CellQuest Pro software, BD Biosciences). Antibodies for flow cytometry analysis were purchased from eBiosciences, Hatfield, UK. Matched isotype controls were used for each antibody to determine gates. Flowjo (Treestar, Ashland, OR) software was used for the analysis of flow cytometry data. Relative PD-L1 expression was expressed as [(MFIPD-L1 siRNA treated sample − MFIisotype)/MFIscrambled siRNA treated sample − MFIisotype)] × 100%, where MFI represents geometric mean fluorescent intensity. Results are expressed as mean ± standard error of mean (SEM) of four independent experiments. Confocal Imaging: SKOV-3-Luc cells were seeded on 13 mm diameter glass cover slips at a seeding density of 3 × 104 cells per slide and treated with PEI–FA/siRNA complexes as described above. Three days post transfection, the cells were fixed with 2% paraformaldehyde for 7 min, washed three times with phosphate-buffered saline (PBS) containing 1% FBS and stained with mouse anti-human PD-L1 primary antibody (eBiosciences, Hatfield, UK). The cells were washed three times and stained with Alexa Fluor® 488 Goat anti-mouse secondary antibody (Life Technologies, Paisley, UK). The cells were washed five times with PBS, mounted on a glass slide using ProLong Gold Antifade Mountant (Life Technologies, Paisley, UK) and imaged using the Leica TCS SP5 Confocal Laser Scanning Microscope. Western Blot: SKOV-3-Luc cells were seeded in 6 well plates at 4 × 105 cells per well and transfected with siRNA as described above. Cells were lysed 3 days post transfection using the RIPA lysis buffer (Santa Cruz Biotechnology, Heidelberg, Germany) and samples were run through a 12% sodium dodecyl sulfate (SDS) polyacrylamide gel. Polyclonal goat anti-PD-L1 antibody (R&D systems, Abingdon, UK), polyclonal rabbit anti-calnexin antibody (Enzo Life Sciences, Exeter, UK) and horseradish peroxidase-conjugated secondary antibodies (Dako, Cambridgeshire, UK) were used for western blotting. Real-Time Quantitative Polymerase Chain Reaction (rt-qPCR): SKOV3-Luc cells were seeded and transfected with siRNA as described earlier. RNA was extracted, purified and converted to cDNA at defined times post transfection using the RNeasy kit and SuperScript III first-strand



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were purchased from Thermo Fisher Scientific, St Leon-Rot, Germany and Qiagen, Manchester, UK, respectively. The human IFN-γ ELISA kit was purchased from eBiosciences, Hatfield, UK. SYBR green PCR master mix, qPCR primers (PD-L1: sense 5′-TATGGTGGTGCCGACTACAA-3′, antisense 5′-TGCTTGTCCAGATGACTTCG-3′; β-Actin: sense 5′-GCTCGTCGTCGACAACGGCTC-3′, antisense 5′-CAAACATGATCTGGGTCATCTTCTC-3′), SuperScript III first-strand cDNA synthesis kit, Dynabeads CD3/CD28 and ProLong Gold Antifade Mountant were purchased from Life Technologies, Paisley, UK. RNeasy mini kit was purchased from Qiagen. Retronectin and Cytokines (IL-2 and IL-4) were purchased from Takara Bio Europe, Saint-Germain-en Lye, France and Peprotech, London, UK, respectively. All other reagents were purchased from Sigma-Aldrich and used as received. The firefly luciferase-expressing SKOV-3-luc-D3 cell line (SKOV3-Luc) was purchased from Caliper (PerkinElmer, Waltham, MA, USA) and grown in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 5% L-glutamine-penicillin-streptomycin solution (Sigma-Aldrich). Primary T cells were grown in T cell medium (RPMI-1640 supplemented with 10% human serum, 5% L-glutamine-penicillin-streptomycin solution (SigmaAldrich) and IL-2, 100 IU per mL or IL-4, 100 IU per mL). PG13 retroviral packaging cells were grown in Dulbecco’s modified eagle's medium (DMEM, Sigma-Aldrich) supplemented with 10% FBS, 5% L-glutamine-penicillinstreptomycin solution (Sigma-Aldrich). Synthesis of Functional Methylcarboxytrimethylene Carbonate (MTC) Monomers (Figure S1, Supporting Information): Synthesis of MTC–SSPEG: MTC–OCH2CH2CH2SS(2-Py) (0.164 g, 0.5 mmol), was first synthesized according to the method reported previously,[26,53,54] and reacted with heterobifunctionalized PEG with thiol and hydroxyl end groups (HS– PEG–OH, Mn 5000 Da, 0.5 g, 0.1 mmol), by dissolving in acetonitrile (2 mL) followed by addition of acetic acid (20 µL). The mixture was stirred overnight and concentrated to dryness. The residue was purified on a Sephadex LH-20 column using tetrahydrofuran (THF) as eluent. MTC–SSPEG was obtained as a white solid (0.59 g, 97%). 1H NMR (400 MHz, CDCl3, 22 °C): δ 4.69 (d, 2H, –CH2OCOO–), 4.30 (t, 2H, – COOCH2–), 4.21 (d, 2H, –CH2OCOO–), 3.63 (s, 455H, H of PEG), 2.96 (t, 2H, –CH2SS–), 2.73 (t, 2H, –SSCH2CH2C(O)NH–PEG), 2.60 (t, 2H, –SSCH2CH2C(O)NH–PEG), 2.08 (m, 2H, –CH2CH2SS–), 1.32 (s, 3H, –CH3). Synthesis of MTC–SSPEG–FA: In a glovebox, MTC–SSPEG (0.33g, 0.066 mmol) and folic acid (FA, 87.4 mg, 0.198 mmol) were dissolved in dry dimethyl sulfoxide (DMSO) (4 mL) and reacted with N,N′dicyclohexylcarbodiimide (DCC, 54.4 mg, 0.264 mmol), dissolved in dry DMSO (1 mL), for 48 h. The solution was dialyzed against DMSO using a dialysis membrane with molecular cut-off (MWCO) of 1 kDa (Spectra/ Por 7, Spectrum Laboratories Inc.) for 48 h, and then precipitated in a mixture of THF and Et2O (1:3) and washed 3 times. MTC–SSPEG–FA was obtained as a yellow solid (0.32 g, 93%) by drying in vacuo. 1H NMR (400 MHz, DMSO-d6, 22 °C): δ 8.64 (d, 1H, H-a), 8.00 (t, 1H, H-e), 7.52 (m, 2H, H-b), 6.92 (m, 1H, H-d), 6.61 (m, 2H, H-c), 4.58 (m, 3H, H-g and H-l), 4.55 (m, 2H, H-f), 4.37 (d, 2H, H-l), 4.21 (t, 2H, H-n), 3.20 (m, 2H, H-p), 2.88 (t, 2H, H-q), 2.78 (t, 2H, H-r), 2.33 (m, 1H, H-j), 1.60–2.00 (m, 4H, H-k, and H-o), 1.18 (s, 3H, –CH3) (Figure S2, Supporting Information). Modification of PEI(Scheme 1): Synthesis of PEI–PEG and PEI– PEG–FA: MTC–SSPEG (76.0 mg, 0.2 mmol) or MTC–SSPEG–FA (163.9 mg, 0.2 mmol), dissolved in dry DCM (2 mL), was reacted with PEI (2g, 0.2 mmol based on 1 mol = 10 000 g = Mn), dissolved in dry DCM (4 mL), for 2 h. The polymers were purified by dialysis against DMSO and then water over 3 days and freeze dried. PEI–PEG, 1H NMR (400 MHz, MeOD, 22 °C): δ 3.62 (s, 455H, H of PEG), 2.58–2.83 (m, br, 930H, H of PEI) (Figure S3, Supporting Information). PEI–PEG–FA, 1 H NMR (400 MHz, D2O, 22 °C): δ 8.57 (s, 1H, H-a), 7.63 (d, 2H, H-b), 6.78 (d, 2H, H-c), 3.63 (s, 500H, H of PEG), 2.63 (m, 930H, H of PEI) (Scheme 1B). Synthesis of PEI–FA: Folic acid (176.6 mg, 0.4 mmol), dissolved in MES buffer (pH 6.0, 3 mL), was reacted with sulfo-NHS (173.7 mg, 0.8 mmol) and EDC (153.4 mg, 0.8 mmol) which were added sequentially. The solution was stirred at 55 °C for 2 h, filtered and added drop wise to PEI (2 g, 0.2 mmol) dissolved in 2-(N-morpholino)ethanesulfonic acid

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www.MaterialsViews.com cDNA synthesis kit, according to manufacturer's instructions. PCR was performed using StepOnePlus Real-Time PCR System (Applied Biosystems, Paisley, UK) and analysed using the 2(-delta delta C(T)) method.[55] Results are expressed as mean ± SEM of three independent experiments. Selective Uptake Studies: PBMCs were isolated using Ficoll density gradient from whole blood. The PBMCs were added to CFSE labelled SKOV-3-Luc cells (1:1 ratio) and left on ice. Polymer/Alexa Fluor 488 tagged siRNA complexes (N/P 50) were added to the cells and incubated at 37 °C for 30 min. Un-internalized siRNA was quenched with trypan blue and cells were washed extensively with PBS before analysis by flow cytometry. SKOV-3-Luc tumour cells and PBMC populations were analysed by gating on CFSE-positive and negative populations respectively. The MFI, which represents the average amount of siRNA that has entered a given cell in the population studied, was analysed by Flowjo. Results represent mean ± SEM of three independent experiments. Retroviral Transduction of Primary Human T Cells: Written informed consent was obtained from participants before enrolling them in the study which was approved by the Hammersmith and Queen Charlotte Hospital's Research Ethics Committee. Peripheral blood was obtained from healthy volunteers for the generation of T4 cells, which are primary human T cells retrovirally (scFv(G250)-CD4TM-γ retroviral vector) transduced to express chimeric cytokine receptors T1E28z and 4αβ in equimolar amounts, according to the previously recorded protocols.[56,57] The PG13 retroviral packaging cells were used to produce the retrovirus into conditioned cell culture medium as described previously.[58] Co-Culture Studies of Tumour and T4 Cells: SKOV-3-Luc cells were seeded and treated with polymer/siRNA complexes as described earlier. At 72 h post transfection, functional grade mouse anti-human PD-L1 antibody (MIH1 clone) or control antibody (50 µg mL−1) was added to the cell culture medium and incubated at 37 °C for 2 h. Antibodies for functional PD-L1 blocking experiments were purchased from eBiosciences, Hatfield, UK. T4 cells were added to tumour cells (1:1 ratio) and incubated for defined periods of time. After co-culture, T4 cells were isolated from the cell culture medium by centrifugation and analysed for CD107a expression by flow cytometry. The conditioned medium was stored at −20 °C for cytokine analysis. The viability of SKOV-3-Luc cells was analysed by the MTT assay. Results for both CD107a expression and cell viability were expressed as mean ± SEM of three independent experiments. Protein Analysis: Conditioned cell culture medium was analysed for human IFN-γ using the human IFN-γ ELISA kit (eBiosciences, Hatfield, UK) according to manufacturer's protocol. Results were expressed as mean ± SEM of three independent experiments. Statistical Analysis: Statistical analysis was carried out using Prism GraphPad 5 (GraphPad, La Jolla, USA) software using Student’s t-test.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements The authors would like to acknowledge the financial support from the Section of Surgery and Cancer, Department of Medicine, Imperial College London and the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore), the Association for International Cancer Research and IBM Almaden Research Center, USA. A*STAR Graduate Scholarship (overseas) from Agency for Science, Technology and Research, Singapore to Pei Yun Teo is gratefully acknowledged. Received: February 7, 2015 Revised: March 17, 2015 Published online: April 11, 2015

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Strategies for cell-mediated immunotherapy of cancer: killing or help?

Effect of targeted ovarian cancer immunotherapy using ovarian cancer stem cell vaccine.

T-cell-targeted immunotherapy.

Hypoxia-targeted siRNA delivery.

Single-walled carbon nanotubes functionalized with aptamer and piperazine-polyethylenimine derivative for targeted siRNA delivery into breast cancer cells.

Delivery of siRNA to ovarian cancer cells using laser-activated carbon nanoparticles.

Efficient siRNA delivery and tumor accumulation mediated by ionically cross-linked folic acid-poly(ethylene glycol)-chitosan oligosaccharide lactate nanoparticles: for the potential targeted ovarian cancer gene therapy.

Beyond adjuvants: immunomodulation strategies to enhance T cell immunity.

Targeted Gene Delivery to MCF-7 Cells Using Peptide-Conjugated Polyethylenimine.

Strategies to genetically engineer T cells for cancer immunotherapy.

Targeted gene delivery to glioblastoma using a C-end rule RGERPPR peptide-functionalised polyethylenimine complex.

Cancer immunotherapy employing an innovative strategy to enhance CD4+ T cell help in the tumor microenvironment.

Cyclodextrin and polyethylenimine functionalized mesoporous silica nanoparticles for delivery of siRNA cancer therapeutics.

Rewiring cancer cell death to enhance oncolytic viro-immunotherapy.

Folic acid-functionalized polyethylenimine superparamagnetic iron oxide nanoparticles as theranostic agents for magnetic resonance imaging and PD-L1 siRNA delivery for gastric cancer.

TLR9-Targeted SiRNA Delivery In Vivo.

Targeted delivery of 5-fluorouracil to HT-29 cells using high efficient folic acid-conjugated nanoparticles.

PDL1 pathway].

Adoptive Engineered T-Cell Trials to Achieve Cancer Killing (ATTACK).

An approach to prepare polyethylenimine functionalized silica-based spheres with small size for siRNA delivery.

Cancer-targeted MDR-1 siRNA delivery using self-cross-linked glycol chitosan nanoparticles to overcome drug resistance.

Cationic Polymer Modified Mesoporous Silica Nanoparticles for Targeted SiRNA Delivery to HER2+ Breast Cancer.

New strategies for cancer immunotherapy: targeting regulatory T cells.

Peptide GE11-Polyethylene Glycol-Polyethylenimine for targeted gene delivery in laryngeal cancer.