Peptide Delivery

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Review

Recent Advances on Anticancer Protein/Peptide Delivery Xun Liu, Fan Wu, Yong Ji, and Lichen Yin Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/ acs.bioconjchem.8b00750 • Publication Date (Web): 14 Nov 2018 Downloaded from http://pubs.acs.org on November 15, 2018

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Bioconjugate Chemistry

Recent Advances on Anticancer Protein/Peptide Delivery Xun Liu 1,#, Fan Wu 1,#, Yong Ji 2,*, Lichen Yin 1,* 1

Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices,

Institute of Functional Nano and Soft Materials (FUNSOM), Collaborative Innovation Center of Suzhou Nano Science & Technology, Soochow University, Suzhou 215123, China 2

Department of Cardiothoracic Surgery, Wuxi People’s Hospital Affiliated to

Nanjing Medical University, Wuxi 214023, China

#

These authors contributed equally.

* Corresponding authors: Email: [email protected] (L. Y.); Phone: 86-512-65882039 [email protected] (Y. J.); 86-510-85351561

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ACS Paragon Plus Environment

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Abstract Protein/peptide drugs possess unique advantages, such as high pharmacological potency, molecular specificity, multifunction, and low toxicity, and thus hold great potentials for cancer therapy. In the past decades, great achievements have been made in protein delivery systems, which can protect cargo proteins against detrimental physiological environments and efficiently deliver proteins into tumor sites and cells. This review first summarized the existing protein/peptide drugs used for cancer treatment, illustrated their anti-tumor mechanisms, and pointed out the potential challenges/barriers against their medical utility. We then discussed the existing strategies for protein encapsulation/conjugation, and then surveyed the recent advances in the development of protein delivery vehicles including lipid-based membrane nanocarriers, polymeric carriers, metal-organic frameworks (MOFs), inorganic carriers, protein/peptide-based nanocarriers, and DNA nanostructures. The design strategies, advantages in potentiating protein delivery efficiencies, and possible limitations of these delivery systems were discussed. Finally, future opportunities and challenges in anti-cancer protein/peptide delivery were indicated.

Keywords: Protein/peptide delivery, nanovehicles, cancer therapy, encapsulation method, protein conjugation, trans-membrane delivery

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Introduction Protein therapeutics has emerged as a crucial strategy for the treatment of cancer, immunological diseases, and metabolic disorders.1 Compared with chemotherapy and gene therapy, protein drugs possess unique advantages in terms of cancer therapy.2 Firstly, protein drugs feature high potency and specificity toward anticancer effect by either directly inducing cancer cell apoptosis via definite signaling pathways, or indirectly inhibiting tumors via modulation of the tumor microenvironment or stimulation of immune response.3, 4 As a result of their potency and specificity, protein drugs usually display notably lower IC50 values than chemodrugs, and are less toxic to normal tissues.5 Meanwhile, protein therapeutics is much less genotoxic than gene therapy, because it is a downstream regulation mechanism that does not alter the genetic makeup.1,6 In addition to protein therapy, peptides with shorter amino acid sequences are also extensively explored and utilized, because of their smaller sizes that are easy to manipulate.7, 8 However, delivering pharmacologically active proteins/peptides to specific tissues or cells encounters various challenges, such as the instability during blood circulation, degradation by enzymes, short half-life, immunogenicity, and inability to cross cell membranes.9 Therefore, various protein delivery systems have been developed to encapsulate proteins, protect them from denaturation and degradation, promote tumor-targeted delivery, enhance the trans-membrane efficiency, and control the protein release/activity in targeted sites.10-12 To this end, proteins are commonly loaded into nanocarriers via physical entrapment or adsorption through hydrogen bond, ionic interaction, and so on.13 Besides, the strategy of chemical modification of proteins/peptides via direct conjugation or covalent encapsulation is also widely explored. Varieties of nanocarriers with multi-functions have thus been developed, which enabled active tumor targeting, deep tumor penetration, and effective cellular uptake. Designing stimuli-labile nanocarriers that are capable of responding to internal physiopathologic characters (redox potential, pH, enzymes, etc) and/or external stimuli (light, magnetic field, temperature, etc), further allowed “on-demand” protein release in the tumor sites or specific subcellular compartment.14, 15 In this review, we summarized the recent advances in anti-cancer protein therapeutics and delivery strategies. As summarized in Figure 1, we first introduced the existing protein/peptides drugs used or explored for cancer treatment and their antitumor mechanism. The different strategies for protein encapsulation or modification were then discussed. We further summarized recent progresses in the development of protein/peptide delivery systems and highlighted their unique functions/applications in cancer treatment. The advantages and limitations of existing design strategies were discussed, which may provide useful insights for future development.

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Figure 1. Scheme of existing anti-cancer protein/peptide therapeutics, major categories of delivery systems, and commonly utilized encapsulation/loading strategies. Proteins/peptides for cancer therapy Protein/peptide therapeutics exploited for cancer therapy mainly include cytokines, antibodies, enzymes, tumor antigens, pro-apoptotic proteins/peptides and other proteins, as summarized in Table 1.

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Table 1 Summary of representative anti-cancer proteins/peptides and delivery systems Types of proteins

Mechanism

Target

Delivery carriers

Cell lines or tumor models

Ref

A549 tumors

16-18

D2F2 cells, D2F2/E2 tumors

17, 19, 20

location Cytokine

IL-2

Activation of NK cells, B cells, cytotoxic T cells and

Extracellular

Nanogels

IL-12

macrophages Stimulate IFN-γ production and induce cytokine

Extracellular

Cell

production of NK cells INF-γ

membrane

coated

nanocarriers

Anti-proliferation, anti-angiogenesis,, stimulation of NK

Extracellular

cells, macrophages, and neutrophils

Nanogels,

OVCAR-3

cells,

protein/peptide-based

OVCAR-3 tumors

A549

and

17, 18, 21, 22

nanocarriers TNF-α

Cytotoxicity on cancer cells and stimulate anti-tumor

Extracellular

immune responses TRAIL

Bind to the death receptors (DR4, DR5) on cell surface

Extracellular

Protein/peptide-based

WEHI-164

nanocarriers

tumors

Cell

COLO-205,

membrane-coated

nanocarriers, nanogels, Antibody

Trastuzumab

Antagonist of HER2-positive breast cancer

Extracellular

micelles, GO,

CNTs,

HUH,

and

cells, A549,

WEHI-164 SK-Hep1,

HepG2

17, 23, 24 25-32

cells,

COLO-205, MDA-MB-231, and

magnetic nanoparticles

A549 tumors

Carrier free

HER2-positive breast, gastric, or gastro-oesophageal

5, 33

junction

carcinoma Pertuzumab

Block heterodimerization of HER2 and HER3

Extracellular

Carrier free

HER2-positive metastatic breast

34, 35

Bevacizumab

Bind and sequester VEGF-A, slow the formation of

Extracellular

Carrier free

Colon tumors, non-small cell lung

35, 36

tumor vasculature Rituximab

tumors, glioblastoma

Inhibit immunosuppressive cytokine (IL-10 and Bcl-2)

Extracellular

Carrier free

expression

Chronic tumors

5


lymphocytic

leukemia

35, 37

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Enzyme

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Atezolizumab

Inhibit PD-L1

Extracellular

Carrier free

Multiple solid tumors

38, 39

Durvalumab

Inhibit PD-L1

Extracellular

Carrier free


40, 41

Pidilizumab

Inhibit PD-1

Extracellular

Carrier free

Hematologic malignancies

39, 42

Nivolumab

Inhibit PD-1

Extracellular

Carrier free


43, 44

Pembrolizumab

Inhibit PD-1

Extracellular

Carrier free


43, 45

Ipilimmab

Block the activity of CTLA4

Extracellular

Carrier free

Melanoma

43, 46

Caspase-3

Catalyze cleavage of cellular proteins to induce

Intracellular

Nanogels,

HeLa, MCF-7, and U-87 MG

47-50

apoptosis Recombinase

proteins-based

carriers

cells, HeLa tumors

Gene modification

Intracellular

DNA tetrahedron

HeLa tumors

47

Carbohydrate hydrolysis

Intracellular

DNA

HeLa cells, HeLa tumors

47, 51, 52

Acute lymphoblastic leukemia

53, 54

B16F10

55-61

(Cre) β-galactosidase (bGal)

tetrahedron,

dendrimers

L-asparaginase

Deplete asparagine

Intracellular

PEGylation

RNase A

Catalyze RNA degradation

Intracellular

Liposomes, MSNs,

nanogels, nanocomplexes,

protein/peptide-based

and

HeLa

cells,

MDA-MB-231, HeLa, and 4T1 tumors

nanocarriers DNase I

Catalyze DNA degradation

Intracellular

Nanogels

A549 tumors

62

Lysozyme

Hydrolyze β-1,4 glycosidic bonds in peptidoglycan;

Intracellular


Sarcoma 180 cells, Hepatoma 22

63-65

nanocarriers

tumors

MSNs, nanogels

U87 MG and C8161 tumors

66-69

MOFs

MDA-MB-231 tumors

70, 71

marine lysozyme inhibits angiogenesis; Egg white lysozyme increases GOx Gelonin

CD8+

T cell number

Catalyze glycose decomposition, producing gluconic

Extracellular

acid and H2O2

/intracellular

Trigger cell apoptosis by cleaving glycosidic bond in

Intracellular

rRNA and disrupting protein synthesis

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cGAMP

A cytosolic DNA sensor catalyzing the synthesis of

synthase

cGAMP that binds and activates STING to induce type I

Intracellular

None

B16F10 cells, B16F10 tumors

Intracellular

Polymersomes, nanogels

H460,

72

IFNs and other immune modulatory molecules Granzyme B

A serine protease cleaving and activating multiple members of the caspase family

Cas9:single-gui de

SKOV3,

and

73-77

MDA-MB-231 tumors

Genome editing against tumor-associated genes

Intracellular

Liposomes

HeLa-DsRed cells

78

Catalyze H2O2 decomposition into O2, which is

Intracellular/

LbLs,

U87-MG, U7-MG tumors, 4T1

79-81

beneficial for radiotherapy and photodynamic therapy

extracellular

polymersomes

tumors

Digest hyaluronic acid

Extracellular

Nanogels, exosomes

A549 and PC3 cells, A549 tumors

(sg)RNA

complexes Catalase

Hyaluronidase

hydrogels,

62, 82

and PC3 tumors

Tumor

Collagenase

Decompose collagen

Extracellular

Gold nanoparticles

OVA

Non-specific antigen

Intracellular

Liposomes,

antigen

micelles,

CPPs mediated carriers

A549 cells

83

E.G7-OVA, B16-OVA, B16F10,

84-86

Mc38, and TC-1 tumors, dendritic cells

TRP2

Tumor-specific antigen, tyrosinase-related protein 2

Intracellular

Liposomes,

MSNs,

gold

Dendritic cell, B16F10 tumors,

87-89

B16F10 tumors

90, 91

nanoparticles Hpg10025-33

Hydrophilic tumor-associated antigenic peptide

Intracellular

Cell

membrane-coated

nanocarriers p-AH1-A5

Tumor-specific antigen peptide

Intracellular

Liposomes

CT-26FL3 RFP/Luc tumors

92

NY-ESO-1

Cancer Testis Antigen

Intracellular

CNTs

B16F10 tumors

93

Melanoma antigen family A

Intracellular


A357 and MCF-7 cells

94

(SLLMWITQV) MAGE-A3

7


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nanocarriers Pro-apopt

p53

otic

Product of tumor suppressor gene TP53, promoting

Intracellular

apoptosis of aberrant cells

Exosomes/vesicles,

MDA-MB-231,

dendrimers, nanogels

and p53-null H1299 cells, HeLa,

protein/pe ptide

SKOV3,

HFF,

14, 95-97

and PC-9 tumors Cytochrome c

Released from mitochondria to induce apoptosis

Intracellular

(caspase pathways) KLAKLAKKL

Liposomes,

MOFs,

H460 and MDA-MB-231 tumors

polymersomes, nanogels

Induce mitochondrial swelling and apoptosis

Intracellular

CPPs-based nanocarriers

AKLAKGG

73, 74, 77, 98-101

HeLa, MCF-7, and HepG2 cells,

102

MCF-7/Adr and MCF-7 tumors

BIM peptide

Bind to all six prosurvival Bcl-2 proteins

Intracellular

Micelles

SKOV3 cells

103

CP11 (cyclic)

Block the phosphorylation of protein kinase CK2

Intracellular

Dendrimers

HeLa cells, MDA-MB-231 tumors

51, 52

PTEN

A tumor suppressor protein as an antagonist of

Intracellular

Liposomes

PC-3 cells

104, 105 52, 106

phosphatidylinositol-3,4,5-triphosphate (PIP3) signaling

Others

AVPIAQK

Induce apoptosis

Intracellular

Dendrimers

HeLa cells

Saporin

Block protein synthesis by depurinating a specific

Intracellular

Polymersomes, dendrimers

HeLa,

nucleotide in the 28S subunit of ribosomes SIRPα

Block interactions between tumor-CD47 and phagocytic

PC-9,

U-87MG,

and

51, 52, 56,

MDA-MB-231 tumors

107, 108

Extracellular

Exosomes/vesicles

HT29 and CT26.CL25 tumors

109

cell-SIRPα to increase tumor cell phagocytosis Hirudin

Anti-angiogenesis

Extracellular

Nanogels

MDA-MB-231 tumors

26, 110

DPPA-1

Bind PD-L1with high affinity

Extracellular

Micelles

B16F10 tumors

111

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Cytokines. Cytokines are a category of secreted proteins that regulate the growth, differentiation, and activation of immune cells via cell signaling.112 For cancer therapy, cytokines could directly induce tumor cell apoptosis or indirectly kill tumor cells by regulating immune responses.5 To date, a variety of cytokines including interleukins (ILs), interferons (INFs), and tumor necrosis factors (TNFs) have been applied in cancer treatment. Interleukin-2 (IL-2), conferring multiple roles as an effector, Treg cell growth factor, and activator of NK cells, B cells, cytotoxic T cells, and macrophages, has been approved for systemic treatment of advanced renal cancer and melanoma.17 Interleukin-12 (IL-12) has emerged as one of the most potent agents for anti-tumor immunotherapy due to its central role in T cell- and NK cell-mediated inflammatory responses.113 For instance, a tumor-targeted oncolytic adenovirus (Ad-TD) was used to deliver non-secreting (ns) IL-12 to tumor cells.19 After intraperitoneal administration of Ad-TD-nsIL-12, the survival of animals bearing orthotopic pancreatic cancer (PaCa) was improved, and peritoneally disseminated PaCa was cured without toxic side effects. Interferon-γ (INF-γ) also exhibits excellent antitumor effect because of its anti-proliferative and anti-angiogenic effect, as well as the ability to stimulate NK cells, macrophages, and neutrophils.17 TNF contains two homologous proteins primarily derived from mononuclear phagocytes (TNF-α) and lymphocytes (TNF-β), which induce antitumor effect mainly through direct cytotoxic effects on cancer cells and modulating antitumor immune responses.5 Joen et al reported a fusion protein consisting of mouse tumor necrosis factor (mTNFα), a linker, and an aptide specific to extra domain B (EDB) of fibronectin (APTEDB), which afforded greater antitumor efficacy than mTNFα alone or mTNFα linked to a non-relevant aptide.23 Despite their antitumor potency, systemic administration of IL-2, IL-12, INF-γ, and TNF at high dose will cause serious side effects.5, 22, 114, 115 One exception is tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the TNF family that mediates tumor cell-specific apoptosis with minimal toxicity toward normal cells. The main anticancer mechanism of TRAIL is binding with death receptor 4 (DR4) and death receptor 5 (DR5).116 Antibodies. Antibodies provoke effective and selective antitumor effect by specifically targeting oncogenic proteins or survival factors on tumor cell surfaces, which represent one of the most successful approaches for cancer treatment.5,35 The mechanisms of antibodies against tumors are varied. Firstly, antibodies directly act on tumor cells, leading to apoptosis or blocking downstream signaling. Monoclonal antibodies against tumors expressing human epidermal growth factor receptor-2 (HER2), vascular endothelial growth factor (EGFR), and CD20 have been widely utilized to achieve remarkable antitumor activity.35 For instance, antagonist trastuzumab is approved for the treatment of HER2-postive breast cancer.5,33 Pertuzumab is another antibody approved by FDA as first-line treatment of HER2-positive breast cancer in combination with trastuzumab and docetaxel.34 Bevacizumab exerts antitumor effect by binding and sequestering EGFR-A and then functionally altering or slowing the formation of tumor vasculature.35, 36 Anti-CD20 antibody rituximab induces remission of low-grade-B-cell lymphoma through antibody-dependent cell-mediated cytotoxicity.35 Secondly, antibody-drug conjugates (ADC) are developed to achieve synergistic antitumor effect. In this strategy, the antibody as targeting ligand could improve the tumor selectivity, enhance drug accumulation in the tumor site, and promote tumor cell internalization. Meanwhile, antibody could induce cytotoxicity by inhibiting or activating particular signaling pathway.5 Recently, FDA has approved two ADCs, brentuximab 9



vedotin in 2011 for the treatment of relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma and ado-trastuzumab emtansine in 2013 for HER2-positive metastatic breast cancer.117 Brentuximab vedotin is a CD30-directed ADC, consisting of the chimeric anti-CD30 IgG1 antibody, the micro tubule-disrupting agent monomethyl auristatin E, and a protease-cleavable linker.118 Ado-trastuzumab emtansine is composed of trastuzumab, DM1, and a stable thioether linker.119 In addition, antibodies could also be conjugated with other proteins. Immunotoxins are recombinant proteins consisting of an antibody or antibody fragment linked to protein toxins such as diphtheria toxin or pseudomonas exotoxin A.120, 121 Denileukin diftitox is approved by FDA for the treatment of CD25-positive cutaneous T-cell lymphoma.122, 123 Finally, antibodies related to immune-mediated pathway have obtained significant clinical attention, such as immune checkpoint blocking antibodies. Cytotoxic T-cell lymphocyte-associated protein 4 (CTLA-4) binds to CD80/CD86 on the surface of antigen-presenting cells (APCs) to limit the activation and proliferation of T cells after recognizing their cognate antigen.124 Ipilimmab as an anti-CTLA-4 antibody was initially approved by FDA in 2011 for the treatment of melanoma.43, 46 However, subsequent research showed that only less than 30% patients with melanoma can get long-term benefit from Ipilimmab therapy and it has a high-grade toxicity.125, 126 Another FDA-approved antibody is the immune checkpoint inhibitor of programmed death receptor 1 (PD-1) or its ligand (PD-L1). PD-1 is expressed on a variety of immune cells and PD-L1 is expressed on tumor cells and APCs.39 The engagement of PD-1 with PD-L1 leads to T cell dysfunction, exhaustion, and interleukin-10 (IL-10) production in the tumor. PD-1 and PD-L1 antibodies are designed to block either PD-1 or PD-L1 and turn on T-cell-mediated immunity. Atezolizumab and Durvalumab, the anti-PD-L1 mAb, show good antitumor activity in multiple solid cancers.38, 40 The anti-PD-1 mAb, Nivolumab and Pembrolizumab, demonstrate antitumor activity in some solid cancers,44, 45 while Pidilizumab displays potential clinical effect in various hematologic malignancies.42 Enzymes. Enzymes have attracted great interests as therapeutics in medical applications due to its highly specific activities and diversity. Up to now, approximately 15% of FDA-approved proteins are enzymes,127, 128 and various enzymes, especially caspase-3, RNase, granzyme, glucose oxidase (GOx), asparaginase, and clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (CRISPR/Cas9) have been exploited for cancer therapy. Since most therapeutic targets of anticancer enzymes are localized inside the cells, the trans-membrane delivery of enzymes into the cytosol is critical toward their therapeutic efficacy. Caspase-3 as a frequently activated death protease, is a crucial mediator of apoptosis, which catalyzes the cleavage of various key cellular proteins.47, 48 RNase mediates cytotoxicity to tumor cells due to cleavage of RNA. RNase A derived from cow pancreas and onconase derived from Northern leopard frog have been used in clinical trials as cancer therapeutics.55,57 Granzyme B is a caspase-like serine protease released by cytotoxic lymphocytes, which could process an extensive array of cell death-related substrates in the cytosol to trigger cell death.76 Therefore, granzyme B could kill tumor cells in diverse ways, such as activating caspases and cleaving key structural proteins in the nuclear membrane or cytoskeleton.129-131 GOx is a widely distributed endogenous glycoprotein consisting of two identical polypeptide chain subunits with two non-covalently bound flavin adenine dinucleotides (FAD) coenzymes.132 GOx could specially catalyze β-D-glucose into gluconic acid and hydrogen peroxide (H2O2), which provides an alternative strategy for cancer starvation therapy. At the meantime, H2O2 is generated at high concentrations 10


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to induce apoptosis of tumor cells.68 L-asparaginase could hydrolyze asparagine into aspartic acid, wherein asparagine is an amino acid essential for tumor growth.53 The CRISPR/Cas9 system as a promising genome editing technology is composed of RNA-guided DNA endonuclease Cas9 and a chimeric single guide RNA (sgRNA).133 Precise genome editing of aberrations in the tumor cells through CRISPR-Cas9 system has been developed for cancer therapy.78 Meanwhile, immune cells edited with CRISPR/Cas9 can also be used for cancer immunotherapies.134 Apart from anti-cancer therapeutic enzymes, some enzymes can exert complementary effect to assist the nano-therapeutics although they do not have antitumor effect. For instance, catalase catalyzes H2O2 into O2, which is beneficial to photodynamic therapy and radiotherapy.79,81 In the tumor interstitium, the extracellular matrix is composed of fibrous proteins and glycosaminoglycans such as collagen, proteoglycan, and hyaluronic acid (HA), which elevate the interstitial pressure and hinder the penetration of nanomedicine. Hyaluronidase and collagenase that can specifically degrade HA and collagen, respectively, could efficiently modify the tumor microenvironment to promote tumor penetration of nanocarriers.62, 135 However, enzyme-based cancer therapy is limited as an adjunctive modality to render tumor cells more susceptible to chemotherapy or immunotherapy. At present, only L-asparaginase is approved by FDA for acute lymphoblastic leukemia (ALL) treatment.128 Moreover, efficiently permeating tumor cell membranes and reducing nonspecific side effect to normal cells are other major challenges. Encapsulation of proteins/peptides into nanostructures would facilitate the cellular uptake, and utilization of cationic materials can further enhance the binding affinity with negatively charged cell membranes to potentiate cellular internalization.56,58 Moreover, amine-rich polymers usually have the “proton sponge” effect to promote endolysosomal escape of protein cargoes.59 Alternatively, cell penetration peptides (CPPs) with potent membrane activities are commonly utilized to promote the trans-membrane delivery of protein cargoes.84, 102, 136 In terms of cancer selectivity, stimuli-responsive nanocarriers are extensively developed and explored to allow cancer-specific release of protein cargoes, such that the undesired toxicity to normal cells could be minimized. Alternatively, proteins could be reversibly caged, such that they are de-activated in normal cells while transform to the activated state in cancer cells upon external or internal stimuli.15, 56, 59, 63 Tumor antigens for immunotherapy. Tumor antigens are mainly represented by tumor-associated antigens (TAA) and tumor-specific antigens (TSA).137 TAA can be proteins or glycoproteins with higher expression levels in tumor cells than in normal cells, which could mark the tumor cell as “others” and thus activate immune response.138 Another category of TAA is cancer-testis antigens (CTA) widely expressed during fetal development but silenced via methylation of the genes in adult tissues, except in germ cells. However, they can be re-expressed in tumor cells due to dysregulated demethylation in tumor cells.139, 140 The tissue-restricted expression profile of CTA has made these antigens as attractive substrates for cancer vaccines. MAGE family proteins and NY-ESO-1 as representative TAA have been widely exploited for caner immunotherapy.140, 141 TSA uniquely expressed by tumor cells belong to the class of neoantigens, which include mutated antigens arising as a consequence of tumor specific DNA damage and viral proteins expressed in virus-induced cancers.138 Usually, tumor antigens are devoured and then processed by APCs, such as macrophages and dendritic cells (DCs). Subsequently, cytotoxic T lymphocytes (CTLs) are activated by antigen presentation and migrated into tumor environment to specifically kill tumor cells.142, 143 As such, efficient delivery of antigens into APCs and reduction of side effects are the major challenges. In addition, 11



administration of tumor antigens only may lead to immune tolerance due to lack of “danger signals” to APCs.144 Co-delivering tumor antigens and adjuvants (such as poly I:CLC as TLR3 ligand, CpG as TLR9 ligand) not only protect tumor antigens against degradation, but also promote uptake by APCs and increase the expression of MHC class I/II molecules. Some nanomaterials inherently have immune-activities in promoting antigen presentation and stimulating immune responses. For instance, Luo et al reported PC7A nanoparticles that induced potent OVA-specific splenocytes killings.86 Usually, nanoparticles with sizes < 500 nm and positive surface charges could be efficiently internalized by DCs. In order to increase DCs-specific delivery and reduce side effect, surface modification of nanoparticles with DC-specific ligands is commonly adopted. Various ligands toward DCs-specific receptors including Fc receptors (FcRs) and a range of C-type lectin receptors (CLRs) have been incorporated into vaccine design.145 Pro-apoptotic proteins/peptides. Apoptosis works like a “suicide” program which involves receptor-mediated pathway (extrinsic) and mitochondria-mediated pathway (intrinsic).146 Receptor-mediated pathway involves death receptors which bind to ligand (such as TRAIL) in the extracellular domain and transmits the death signal from the surface to the intracellular signaling pathways. Mitochondria-mediated pathway is activated due to the change of mitochondrial membrane permeability, which results in the loss of the mitochondrial transmembrane potential and the release of pro-apoptotic proteins. The pro-apoptotic proteins/peptides could activate the caspase-dependent pathway or may be apoptosis-inducing factors.146 p53 protein is a transcription factor as the product of tumor suppressor gene p53, which can promote apoptosis of aberrant cells through both transcription-dependent and -independent mechanisms.147 Nearly 50% of human tumors have mutant p53 proteins.14 Cytochrome c is an intermembrane space protein in the mitochondria, and cytochrome c-initiated pathway is the major caspase activation pathway. Cytochrome c released from mitochondria can induce a series of biochemical reactions that result in caspase activation and subsequent cell death, which represents the major caspase activation pathway.73, 99 Challenges of intracellular delivery are also posed for most pro-apoptotic proteins because they function inside the cancer cells. Kim et al conjugated cytochrome c with a membrane permeable sequence (MPS) peptide and incorporated it into DOPE/DOTAP nanoparticles to facilitate the trans-membrane delivery.98 Gao et al conjugated a cell penetrating peptide, cyclosporin A, with a pro-apoptotic peptide to improve its internalization efficiency.102 Others. An increasing number of protein drugs are being discovered, which can kill tumor cells by other mechanisms. Saporin is a 30-kDa, positively charged and membrane-impermeable ribosome inactivating protein (RIP), which could irreversibly inhibit protein synthesis in eukaryotic cells by rending the subunit of ribosomes.51, 56 Saporin is used in clinical trials in patients refractory to traditional chemotherapy.56 SIRPα is a regulatory membrane glycoprotein expressed mainly by myeloid cells. CD47 is the receptor of SIRPα, which is a transmembrane protein commonly upregulated on the surface of tumor cells. The interaction of SIRPα on phagocytic cells with CD47 can inhibit their phagocytic function, serving as a “don't eat me” signal.109 Blockage of SIRPα-CD47 signaling by using anti-SIRPα antibodies or recombinant SIRPα as competitive antagonists of CD47 could directly induce phagocytosis of macrophages and elicit immunotherapeutic outcomes.109, 148 Strategies for protein modification and encapsulation Covalent conjugation. The strategy of conjugating protein drugs to polymers has been well 12


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developed in the past decades. The functional groups on the surface of proteins including carboxylic acid, amine, hydroxyl, and thiol are utilized to bind to polymers such as PEG, and the obtained prodrug features prolonged blood circulation and enhanced tumor accumulation.1, 149 Some of them have been approved by FDA for clinical use or are under clinical investigation.54 Particularly, PEGylation inhibits protein aggregation/fibrosis, reduces protein clearance, inhibits enzymatic degradation, and provides an effective shield against the immune system. For example, the half-lives of IFN-2a and -2b are below 12 hours, while PEGylated IFN has a substantially increased serum half-life (48–72 h).150 Native L-asparaginase causes hypersensitivity reactions and shock, while PEGylated L-asparaginase shows increased serum half-life with lower occurrence of hypersensitivity.53, 54 However, PEG-protein conjugates are usually from nonspecific reaction of PEG with proteins, which may lead to heterogeneous mixtures of multiple PEGs attached at different sites, thus reducing the protein bioactivities. To address this issue, specific modification chemistries have been developed. Popp et al. utilized sortase-mediated transpeptidation to facilitate site-specific attachment of PEG to cytokines.151 They showed that cyclized IFN overhanging PEG had greater thermostability and longer circulation half-life. In addition, the hydrophilic PEG corona will reduce the interaction with cancer cell membranes to reduce cancer cell uptake.152 Poly(α-amino acid)s (PαAAs) are promising alternative to PEG for protein conjugation due to their excellent biodegradability and chemical modulability.153 Talelli et al designed a reduction sensitive poly-L-glutamic acid (PGA)-lysozyme conjugate with masked protein activity by reacting pyridyl dithiol modified PGA with N-succinimidyl-S-acetylthiopropionate (SATP) modified lysozyme.63 In the reducing intracellular environment, proteins were released from the conjugates to regain activity. Hou et al describe a one-pot, two-step polymerization process to synthesize site-specific topological protein-poly(amino acid) (PAA) conjugates.153 A phenyl thioester for native chemical ligation (NCL) and a polyglycine for sortase A mediated ligation (SML) were in situ installed at the C- and N-termini of PAAs, respectively, to allow rapid conjugation with various proteins under mild conditions. Particularly, the cyclic conjugates exhibited remarkable thermostability and protease resistance. Furthermore, they reported a head-to-tail IFN-poly(α-amino acid) macrocycle conjugate, circ-P(EG3Glu)20-IFN, and compared the antitumor activity of circ-P(EG3Glu)20-IFN against its linear counterparts (Figure 2).21 They found that circ-P(EG3Glu)20-IFN significantly improved protease resistance, binding affinity, and in vitro anti-proliferative activity toward OVCAR3 cells. Moreover, circ-P(EG3Glu)20-IFN exhibited longer circulation half-life, higher tumor retention, deeper tumor penetration, subsequently inhibiting tumor progression in mice bearing OVCAR3 and SKOV3 xenograft tumors without causing severe paraneoplastic syndromes.

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Figure 2. Synthesis routes for four IFN-polymer conjugates: the N-terminal conjugate N-P(EG3Glu)20-IFN and the macrocyclic conjugate circ-P(EG3Glu)20-IFN (A), the C-terminal conjugate C-IFN-P(EG3Glu)20 (B), and the C-terminal PEG conjugate C-IFN-PEG (C). Reproduced with permission from Ref 21.

Recently, Jiang et al reported a supramolecularly engineered circular bivalent aptamer for functional protein delivery.154 They conjugated β-cyclodextrin onto a single-stranded sgc8 aptamer which could target protein tyrosine kinase-7 (PTK-7), and further cyclized it with a complementary sequence (monoaptc) to form β-cyclodextrin-conjugated circular bivalent aptamers (cb-apt-βCD) Adamantane-modified protein was encapsulated into the cb-apt to form supramolecular assembly via host-gest interaction for enhanced intracellular delivery. Cytotoxic saporin was further used as therapeutic protein to efficiently inhibit HeLa cell growth. Reversible modification of proteins with trigger-cleavable motifs has been developed to precisely and spatiotemporally modulate the protein activity. Deactivated proteins due to chemical protection are reactivated after removal of the blockage moieties by external or cancer-specific internal stimuli, thus enabling selective anti-cancer efficacy. Wang et al modified the lysine residues of proteins (including RNase A and saporin) with cis-aconitic anhydride, which could be removed in the slightly acidic intracellular endolysosomes in cancer cells to reactivate the proteins.56 Furthermore, they developed a RNase A prodrug by modifying its lysine residues with 4-nitrophenyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) benzyl carbonate (NBC).58 The deactivated RNase A–NBC (RNBC) could restore its biological activity by elevated ROS in cancer cells. Considering that the ROS concentration in cancer cells is still not sufficient to induce complete removal of NBC moieties, we recently delivered a cancer-targeting vehicle to co-delivery RNBC and photosensitizer.59 Upon tumor site-specific light irradiation, the photosensitizer greatly enhanced the intracellular ROS concentration to completely restore the 14


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anti-cancer activity of RNase A. Physical encapsulation. Large numbers of materials that form nanocarriers with hydrophilic cavities could encapsulate protein cargoes. For example, liposomes with a bilayer structure and polymersomes with a polymeric membrane can encapsulate hydrophobic molecules within their membranes while load hydrophilic biomolecules within their aqueous cores.85, 108 Mesoporous silica nanoparticles (MSNs) can also easily encapsulate proteins with high molecular weights within their large pore channels.88 The protein/peptide encapsulation efficiency could be further augmented by incorporating additional physical interactions between materials and proteins, such as electrostatic interaction, hydrogen bonding, hydrophobic interaction, and van der Waals force. For example, Yang et al reported anisamide-functionalized bioresponsive chimaeric nanopolymersomes (Anis-BCPs) where a small segment of poly(acrylic acid) (PAA) was incorporated into the hydrophilic segment that faced the internal cavity.74 When positively charged FITC-cytochrome C was used as a protein cargo, its encapsulation efficiency exceeded 96% due to the electrostatic interaction with negatively charged PAA. Nanocapsules formed from hydrophobic polymers such as poly(lactic-co-glycolic acid) (PLGA), are also extensively utilized for protein encapsulation. At present, multiple methods including double emulsion, phase separation, spray-drying, microfluidics, and so on have been applied to encapsulate proteins into polymeric nanocapsules.155 Double emulsion and phase separation are two major methods used, due to their simplicity in controlling particle size. Spay-drying method was developed to overcome scalability issues of emulsion-based methods. Microfluidics with more precise control over process parameters serves as a promising alternative to conventional emulsion-based techniques. However, organic solvent and shear forces during emulsification could induce denaturation, and microfluidics may suffer from difficulties in scale up. Chen et al used PLGA to encapsulate catalase as a O2-evolving agent into the aqueous core of nanoparticles by using the double emulsion method, and the O2 generated disrupted the PLGA membrane to trigger cargo release.79 Pessi et al employed microfluidic technology to encapsulate bovine serum albumin (BSA), and the obtained particles were intact, monodisperse, non-porous, and stable up to 4 weeks.156 Self-assembly. Self-assembly processes involve specific intra- and intermolecular interactions to form functional supramolecular composites.157 Proteins can serve to form nanocomposites with various materials by self-assembly via electrostatic interactions, hydrogen bonding, hydrophobic interactions, and etc. For instance, cationic, ketal-containing polyethylenimine (KPEI) forms binary nanocomplexes with anionic RNBC via electrostatic interactions, and acid-responsive degradation of KPEI in the endolysosomes could facilitate RNBC release.59 Similarly, Wang et al reported a protein delivery platform prepared by combinatorial design of cationic lipid-like materials, named as lipidoids.56 To strengthen the electrostatic assembly, protein drugs such as RNase A and saporin were modified with cis-aconitic anhydride on their lysin residues, such that they would bear more negative charges to interact with positively charged lipidoids. Wang et al reported a new synthetic CPP with cationic charges that can bind to the antigen protein ovalbumin (OVA) via electrostatic self-assembly to form peptide/OVA nanocomposites.84 Chang et al developed a guanidyl modified dendrimer to form nanocomplexes with various proteins/peptide including bovine serum albumin, R-phycoerythrin, p53, saporin, β-galactosidase, and peptides via hydrogen and salt bridge.52 In addition, proteins could be fabricated into fluoroamphiphilic nanoparticles via fluorination effect. Zhang et al reported fluoroalkanes modified 15



polyethylenimines (PEIs) for protein delivery,107 and the fluorous substituents played essential roles in the formation of uniform nanoparticles. Polymerization-based encapsulation. Proteins and monomers are dissolved in water, and the protein-encapsulated nanoparticles are formed during the polymerization of monomers. Microemulsion and inverse microemulsion polymerization are two most common techniques used for protein encapsulation. For instance, Murthy et al. used microemulsion polymerization to prepare OVA-loaded nanoparticles with hexane as the continuous phase, dioctyl sufosuccinate and Brij as the surfactants, and bisacrylamide benzaldehyde acetal as acid-degradable crosslinker.158 The particle size varied between 1 and 10 μm that was appropriate for phagocytosis by APCs. They further reported a more hydrophilic acid-degradable crosslinker containing triglyme moiety, which is more suitable for inverse microemulsion polymerization.159 However, emulsion based polymerization usually uses organic solvent which may deteriorate protein activity. Meanwhile, the shear stress during the formation of microemulsion may destabilize the proteins. To overcome these issues, in situ polymerization was developed for protein encapsulation. Polymerizable vinyl monomers were adsorbed on the surface of proteins via electrostatic interaction or covalently conjugated to the proteins and the polymerization was performed in aqueous solution, forming nanocapsules at the meantime. Using this technique, Gu and co-authors prepared protein nanocapsules by incorporating photo-sensitive peptide as crosslinker to achieve controlled protein release triggered by UV and enzymes.49 Caspase-3 was selected as a model protein and a photo-labile, o-nitrobenzyl ester-modified VDEVDTK bisacrylated peptide was used to crosslink the polymeric network. Similarly, they developed redox-responsive nanocapsules using disulfide-bond containing N,Nˊ-bis(acryloyl)cystamine as crosslinker.50 Acrylamide was further used as a general building block of water-soluble shell, and N-azidodeca(ethylene glycol)ethylacrylamide was selected as another monomer to synthesize a nearly neutral polymer shell to avoid non-specific internalization into normal cells (Figure 3).14 The nanocapsules were crosslinked with N,Nˊ-bis(acryloyl)cystamine to allow redox-responsive protein release in the cytoplasm.

Figure 3. (A) Schematic diagram of clickable, redox-sensitive protein nanocapsules and the conjugation to DBCO TAMRA by copper-free click chemistry. (B) Schematic illustration of LHRH-conjugated nanocapsules. Reproduced with permission from Ref 14. 16


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Protein delivery systems Lipid-based membrane nanocarriers Liposomes. Liposomes are a class of nano/microsized self-assembled vesicles consisting of one or multiple hydrophobic phospholipid bilayers surrounding the aqueous solution core. Up till now, various liposomes including stealth liposomes, stimuli-responsive liposomes, and targeted liposomes have been widely investigated as protein/peptide carriers to enhance cargo stability, prolong systemic circulation, control drug release, enhance tumor accumulation, and reduce adverse side effects.160, 161 Yoshizaki et al reported a type of cationic lipid-incorporated, pH-sensitive polymer-modified liposomes as OVA delivery carriers for cancer immunotherapy.85 Egg yolk phosphatidylcholine (EYPC) liposomes were modified with 3-methylglutarylated hyperbranched poly(glycidol) (MGlu-HPG), which were destabilized below pH 6.0 due to the conversion of hydrophilicity to hydrophobicity. Cationic lipid inclusion improved their pH sensitivity at weakly acidic pH and induced up-regulation of antigen presentation-involved molecules on DCs. Moreover, antigen presentation via MHC class II was promoted by cationic lipid inclusion. Administration of the cationic lipid-incorporated liposomes to mice bearing E.G7-OVA tumor demonstrated stronger anti-tumor efficacy than liposomes without cationic lipids. Noh et al developed a new type of multifaceted immunomodulatory nanoliposomes, named as tumosomes, which could transform tumors into vaccines and induce enhanced antitumor immune response.162 Tumosomes are comprised of tumor cell membrane proteins as tumor-associated antigens, 3-O-desacyl-4′-monophosphoryl lipid A (MPLA) as adjuvant, cationic lipid dimethyldioctadecylammonium bromide (DDA) as cell-invasion moiety, DOPC, and cholesterol. Tumosomes displaying a whole array of tumor-associated antigens greatly diminished the chance of tumor escape. MPLA adjuvant augmented the immunogenicity of purified tumor cell membrane antigens, and the cationic DDA improved the uptake of tumor antigens by APCs. Cell-secreted exosomes/vesicles. Exosomes are composed of lipid bilayer-enclosed, nanosized extracellular vesicles containing proteins and RNA, which are released by most cell types.163 Due to their cellular origin and intrinsic functions of transferring biological information, exosomes have been widely exploited for drug delivery. Hong et al developed an engineered enzymatic exosome with the native glycosylphosphatidylinositol (GPI)-anchored form of hyaluronidase (Exo-PH20) to overcome the immunosuppressive and anticancer therapy-resistant tumor microenvironment.82 They demonstrated that Exo-PH20 could deliver GPI-anchored hyaluronidase to the overly accumulated extracellular matrix (ECM), decompose HA overexpressed in the tumor microenvironment with higher enzymatic activity than recombinant human hyaluronidase, and effectively suppressed tumor growth. Mizrak et al generated genetically engineered cells which stably expressed the suicide therapeutic mRNA/protein for cytosine deaminase (CD) fused in-frame with uracil phosphoribosyltransferase (UPRT).164 The secreted microvesicles (MVs) containing the CD/UPRT fusion enzymes were harvested and used to treat schwannoma tumors. MVs were capable of transferring mRNA/protein to tumor cells, and they significantly inhibited schwannoma tumor growth when combined with 5-fluorocytosine (5-FC). Arrestin domain containing protein 1 [ARRDC1]-mediated microvesicles (ARMMs) are another extracellular vesicles (EVs) distinct from exosomes.165 Endogenous proteins such as cell surface receptors can be actively recruited into ARMMs and delivered into recipient cells to initiate intercellular communication. Therefore, exogenous cargoes could be similarly packaged 17



into ARMMs. Wang et al constructed an ARRDC1-p53 fusion protein that could be incorporated into ARMMs. They demonstrated that ARMMs could efficiently deliver p53 into multiple tissues in vivo.166 Cell membrane-coated nanoparticles. Biomimetic delivery systems based on coating nanocarriers with cell membranes via chemical conjugation or noncovalent binding are being intensively pursued due to various merits including alleviating immunogenicity, enhancing biocompatibility, prolonging circulation, and achieving tumor targeting.167 Typical cell membranes used include red blood cell, white blood cell,168 platelet, and tumor cell membranes.25, 169 Guo et al used erythrocyte membrane-enveloped PLGA nanoparticles for the delivery of antigenic peptide (hgp10025-33) and monophosphoryl lipid (MPLA) to APCs.90 The nanovaccine was further modified with mannose to target APCs in the lymphatic organ, and it effectively inhibited tumor growth and suppressed tumor metastasis. Hu et al developed a platelet membrane-coated core-shell nanovehicle for sequential and site-specific delivery of TRAIL and DOX.25 DOX was encapsulated into the inner core and the outer shell of platelet membrane was employed for decoration of TRAIL. Zhang et al genetically engineered platelets from megakaryocyte (MK) progenitor cells to express PD-1.170 The PD-1 platelet and its derived microparticles could accumulate in the surgical wound of tumors and revert exhausted CD8+ T cells, resulting in the eradication of residual tumor cells. Fang et al reported the biological functionalization of cancer cell membrane-coated PLGA nanoparticles (CCNPs), which had two distinct anticancer modalities, anticancer vaccination and homotypic targeting drug delivery.169 On one hand, CCNPs prepared using B16F10 melanoma cell membrane could efficiently deliver TSAs combined with immunological adjuvants (MPLA) into APCs to promote immune responses. On the other hand, PLGA nanoparticles coated with MDA-MB-435 cancer cell membranes possessing cell adhesion molecules showed significantly increased cellular adhesion to the source cells compared to non-coated PLGA nanoparticles. Polymeric carriers Polymeric micelles. Amphiphilic copolymers could self-assemble into micelles with core-shell structure. Among them, polyionic complex micelles were developed by incorporating neutral and ionic parts, and they were able to encapsulate proteins with counter charges via electrostatic interaction. For example, Luo and co-workers screened a library of ultra-pH-sensitive (UPS) ionizable block copolymer micelles (20-50 nm) containing tertiary amines with linear or cyclic side chains on the hydrophobic segment, and evaluated their abilities in generating a cytotoxic T lymphocyte (CTL) response (Figure 4).86 Ovalbumin (OVA) was selected as model antigen and the OVA loading efficiency was >75%. The OVA-loaded PC7A micelles induced notable OVA-specific splenocytes killings and achieved efficient cytosolic antigen delivery to APCs in draining lymph nodes, and they simultaneously activated the stimulator of interferon gene (STING) pathway. Lin et al reported PEGylated cationic amphiphilic polymers (Alkyl-PEI2k-PEG2k) that formed micellar hybrid nanoparticles to carry TRAIL (IPN@TRAIL).31 Photothermal agent, metalla-aromatics complex Ph556, and iron oxide were encapsulated into the core of the resultant micelles, and negatively charged TRAIL was adsorbed onto the micelles surface through electrostatic interaction. IPN@TRAIL greatly improved synergistic effects between photothermal therapy and TRAIL therapy.

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Figure 4. a) Screening of polymer structures that generate strong OVA-specific CTL response. b) Quantitative comparison of OVA-specific CTL responses. Reproduced with permission from Ref 86.

Polymersomes. Polymersomes composed of block or graft amphiphilic copolymers have similar property to liposomes yet higher stability. Hydrophilic drugs, such as proteins and peptides, could be loaded within their inner cavity. Moreover, it is possible to control the size, shape, membrane thickness, and surface chemistry of polymersomes for precision delivery.171 Via crosslinking of the hydrophobic block, polymersomes could be further stabilized, while incorporating stimuli-responsive domains into the hydrophobic block would render on-demand drug release profiles. To enhance the loading of proteins/peptides into polymersomes, the hydrophilic segments facing the inner cavity can also be modulated to carry different charges, such that the electrostatic interactions with the protein cargo could additionally promote drug encapsulation. For instance, Jiang et al reported an angiopep-2-directed and redox-responsive virus-mimicking polymersomes (ANG-PS) that can efficiently and selectively deliver chaperone saporin (SAP) to orthotopic human glioblastoma xenograft in nude mice.108 ANG-PS was prepared from poly(ethylene glycol)-b-poly(trimethylene carbonate-co-dithiolane trimethylene carbonate)-b-polyethylenimine (PEG-P(TMC-DTC)-PEI, a triblock copolymer, and ANG-functionalized PEG-P(TMC-DTC) (ANG-PEG-P(TMC-DTC), a diblock copolymer. Angiopep-2 was used to target low-density lipoprotein receptor-related protein-1 (LRP-1) on cancer cells, and the reversible cross-linking of the DTC segment via disulfide bonding stabilized the polymersomes. ANG-PS had a size of 76 nm and high SAP loading content of 8.8%, likely due to the hydrogen bonding and ionic interaction between negatively charged SAP and positively charged PEI. More importantly, the disulfide bonding could be cleaved in the cytosol by GSH, and instantaneous SAP release was allowed to induce strong anti-tumor efficacy. Similarly, the redox-responsive, reversibly crosslinked polymersomes were dually functionalized with cRGD and fusogenic GALA peptides for efficient delivery of cytochrome C.100 Robertson et al also reported pH-sensitive tubular polymersomes based on the copolymer 19



poly(2-(methacryloyloxy)ethylphosphorylcholine)-co-poly(2-(diisopropylamino)ethyl methacrylate) (PMPC–PDPA). Fluorescence-labeled BSA was encapsulated into the polymersomes at drug loading of 29%, and its release was initiated at the endolysosomal acidic pH (6.2) due to protonation of its tertiary amine groups that led to disassembly of the polymersomes.172 Layer-by-layer carriers. Layer-by-layer (LbL) system is a multilayered thin film normally obtained based on numerous forces including charge and hydrogen bonding association.173 LbL system has been widely used for biomedical application due to its simplicity and versatility. Through consecutive electrostatic adsorption of oppositely charged polymers, proteins could be encapsulated into the LBL system. A key advantage of the LbL system is the capacity to directly load proteins and other bioactive agents into LbL films for synergistic therapy or multiple clinical indications. Song et al prepared a tantalum oxide (TaOx) nanoshell to encapsulate catalase (Cat) that could decompose H2O2 into O2 and H2O.80 Furthermore, TaOx@Cat nanoshells were coated with cationic polyallylamine hydrochloride and anionic polyacrylic acid via the LbL method, and then conjugated with amine-terminated PEG to enable colloidal stability and long blood circulation. They demonstrated that TaOx@Cat-PEG could efficiently decompose endogenic H2O2 and improve tumor oxygenation. Therefore, a remarkable synergistic radiation therapy sensitization effect was achieved. Dendrimers. Dendrimers are hyperbranched polymers with monodispersity and nanometric size. Due to the controllable architecture and surface groups, dendrimers have been used for drug delivery.174 Via electrostatic interaction, negatively charged proteins are easily composited with positively charged dendrimers, such as PAMAM.51, 59 More importantly, dendrimers could obtain versatile function by surface chemical modification, enabling a “multivalent” structure. Chang et al developed a dendrimer-based polymer consisting of a dendrimer scaffold, a hydrophobic membrane-disruptive region (aromatic motif), and a multivalent protein binding surface (guanidyl) for the efficient delivery of bovine serum albumin, R-phycoerythrin, p53, saporin, β-galactosidase, and peptides into the cytosol of living cells (Figure 5).52 Guanidyl ligand formed strong hydrogen bonds and salt bridges with amides and oxyanions in proteins, and its benefited efficient endocytosis. The integrated phenyl groups promoted endocytosis and facilitated endosomal escape by disrupting endosomal membranes. Lv et al prepared a library of fluorodendrimers to encapsulate proteins, and an efficient and non-toxic fluorodendrimer, A6-2, was identified, which was capable of transporting various proteins including bovine serum albumin, β-galactosidase, saporin, and a cyclic hendecapeptide into the cytosol of living cells.51 Moreover, activities of the delivered proteins/peptides were maintained. Modification of dendrimers with fluoroalkyls or fluoroaromatics improved the protein stability, promoted endocytosis as well as endosomal escape due to the efficient interactions between fluoro-structures and phospholipid bilayers.107 When the A6-2/saporin complexes were coated with a hyaluronic acid shell, they afforded desired serum stability and tumor targeting, and thus exhibited potent antitumor effect in MDA-MB-231 breast tumor models following i.v. injection.

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Figure 5. Dendrimer-based polymer and its features in intracellular protein delivery (A). Proposed mechanism for intracellular protein delivery (B). Reproduced with permission from Ref 52.

Nanogels. Nanogels are nanoscale three-dimensional polymer networks containing a large amount of water. As such, nanogels can easily encapsulate hydrophilic protein/peptides within their matrix. Nanogels can be prepared by physical or chemical cross-linking. Amphiphilic polysaccharides were first used to create physically cross-linked nanogels via hydrophobic interactions in water.175 Chemically cross-linked nanogels were thereafter reported by in situ polymerization or inverse microemulsion polymerization of monomers and cross-linkers.14, 159 Nanogels were also prepared by chemically cross-linking the polyion cores.18, 176 Zhu et al reported a tumor-specific, self-degradable, collaboratively crosslinked nanogel (cNG) for DNase I delivery (D/aH-cNG) (Figure 6).62 The crosslinked nanogel was obtained via the self-assembly and radical polymerization of cholesteryl-6-aminohexylcarbamate methacrylated hyaluronic acid (cm-HA). To enable the tumor-specific self-degradability of the cNG, hyaluronidase was modified and deactivated with citraconic anhydride, and the obtained tumor-acidity-activatabe hyaluronidase (aHAase) was encapsulated into the nanogel. The extracellular slight acidity in tumor tissues activated aHAase to partially degrade HA, which increased the diffusion of cNG into deep tumors. In the acidic endolysosomes, aHAase was completely reactivated to break down the nanogel, releasing the DNase I to kill cancer cells. Chen et al reported in situ-forming, reduction-degradable nanogels based on poly(ethylene glycol)-b-poly(2-(hydroxyethyl) methacrylate-co-acryloyl carbonate) (PEG-P(HEMA-co-AC)) block copolymers.101 The nanogels were formed in the presence of cystamine via ring-opening reaction with cyclic carbonate groups. FITC-labeled cytochrome c could be efficiently encapsulated into the nanogels at drug loading up to 48.2%, and it showed redox-responsive release profiles due to nanogel disassembly. Ding et al reported pH-sensitive coiled-coil peptide-cross-linked hyaluronic acid nanogels (HA-cNGs) obtained from an equivalent mixture of hyaluronic acid conjugates with GY(EIAALEK)3GC (E3) and GY(KIAALKE)3GC (K3) peptides.99 Mild acidic environment greatly accelerated the 21



cytochrome c release.

Figure 6. a) Schematic illustration of self-assembly and tumor-specific self-degradation of the collaboratively crosslinked D/aH-cNG. b) Scheme of enhanced protein delivery by the D/aH-cNG for cancer therapy. Reproduced with permission from Ref 62.

Scaffolds and hydrogels. Scaffolds and hydrogels are three-dimensional polymeric matrix that can swell but not dissolve in water or biological fluids. Because of their biocompatibility and design flexibility, scaffolds and hydrogels based on either synthetic or natural polymers have been widely used for the controlled release of therapeutic drugs, proteins, peptides, and nucleic acids. Particularly, because of the three-dimensional networks with tunable pore size, mechanical strength, and degradation rate, hydrogels are often used to enable localized and sustained cargo release, prolong the retention of protein cargoes in the tumor tissues, and protect proteins against denature in vivo.177 Recently, hydrogels are widely used to realize encapsulation and controlled release of anti-cancer immuno-proteins.81, 178, 179 Wang et al developed an in situ forming, reactive oxygen species (ROS)-responsive scaffold for gemcitabine (GEM) and anti-PD-L1 blocking antibody (aPDL1) combination therapy (Figure 7).178 The scaffold was obtained by crosslinking poly(vinyl aclcohol) (PVA) and N1-(4-boronobenzyl)-N3-(4-boronophenyl)-N1,N1,N3,N3 -tetramethylpropane-1,3-diaminium (TSPBA), and it released GEM and aPDL1 in a programmed manner in response to ROS in the tumor microenvironment. The scaffold elicited an immunogenic tumor phenotype via local GEM delivery, and promoted an immune-mediated tumor regression by local release of aPDL1 in B16F10 melanoma and 4T1 breast tumor models. Moreover, tumor recurrence was prevented after primary resection.

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Figure 7. Schematic illustration of in situ formed ROS-responsive gel scaffold for GEM and aPDL1 combination chemoimmunotherapy. Reproduced with permission from Ref 178.

Yu et al reported a thermos-gelling, ROS-responsive polypeptide gel for sustained release of anti-PD-L1 and dextro-1-methyl tryptophan (D-1MT).179 The gel was formed from a functional triblock copolymer, P(Me-D-1MT)-PEG-P(Me-D-1MT), synthesized via ring-opening polymerization (ROP) of L-methionine (Me) N-carboxyanhydride (NCA) and D-1MT NCA with PEG-NH2 as the initiator. Hydrogel was formed at a high concentration (8.0 wt%) upon rising temperature, and the poly(L-methionine) region conferred H2O2-responsive property through the oxidation of the sulfoether into sulfoxide or sulfone, which promoted the cargo release. The anti-PD-L1-loaded hydrogel exhibited synergistic immune-antitumor efficacy in melanoma-bearing mice. Mental-organic-frameworks (MOFs). MOFs are hybrid polymers consisting of metal ions/cluster and organic ligands, which have been extensively explored for drug delivery because of their non-toxicity, biodegradability, tunable pore scale, selectable composition, large surface area, and good thermal stability.180 Cheng and co-workers encapsulated gelonin, an rRNA disruption N-glycosidase, into pH-responsive MOF nanoparticles formed via self-assembly of blocks of metal nodes (Zn2+) and organic ligands (2-methylimidazole) and further camouflaged with an extracellular vesicle membrane (EVM) derived from MDA-MB-231 cells.70 After uptake by cells, the pH drop from pH 6.0-6.5 in early endosomes to pH 4.5-5.5 in late endosomes and lysosomes lead to the release of the organic ligand of the imidazole derivative, thus enabling buffering effect upon protonation of the imidazole ring. Inorganic vehicles Mesoporous silica nanoparticles (MSNs). MSNs with large pore volumes and surface areas have been widely employed for controlled release of drugs, genes, and proteins. Modifying MSNs with various functional groups via covalent bonding or electrostatic interactions provides the materials with great versatility and mechanical features. More importantly, hydrophilic proteins with high MWs can be easily encapsulated within their large pore channels.181 Recently, Zhu and co-workers reported a mesoporous silicon vector (MSV) containing antigen (tyrosinase-related protein 2 peptide, TRP2 peptide) and dual Toll like receptor (TLR) agonists, CpG, and monophosphoryl lipid A (MPLA), which could induce synergistic antitumor T cell response against B16 melanoma.88 They demonstrated that MSV protected the peptide from rapid degradation and enabled co-delivery of CpG and MPLA along with TRP2 into the same DCs, thus increasing the efficiency of DCs to induce TRP2-specific CD8+ T cell responses. Fan et al 23



designed a hollow mesoporous organosilica nanoparticle (HMON) to co-deliver glucose oxidase (GOx) and L-Arg for synergistic starvation and gas therapy.66 GOx was covalently conjugated onto the surface of HMON and L-Arg was encapsulated into the hollow cavity through hydrogen bonding and electrostatic interaction. GOx catalyzed glucose into gluconic acid and H2O2, and H2O2 oxidized L-Arg into NO and further reacted with NO to produce biocidal peroxynitrite molecules (ONOO-) with stronger anti-tumor efficacy than H2O2. Shao et al fabricated oxidative and redox dual-responsive MSNs containing diselenide-bond for encapsulating RNase A.60 After surface coating with cancer-cell-derived membrane, the RNase A-loaded MSNs displayed increased blood circulation, homologous targeting, enhanced tumor accumulation, and efficient tumor growth inhibition. Gold nanovehicles. Gold nanovehicles have attracted extensive attentions in biomedical applications due to their satisfactory performances including controlled sizes and structures, biocompatibility, and unique optical properties.182 Liang and co-workers designed a versatile nanovaccine based on liposomes-coated gold nanocages (Lipo-AuNCs) modified with DC-specific antibody aCD11c for the targeted delivery of TRP2 peptide and adjuvant MPLA.89 The hydrophobic peptide TRP2 was loaded in the inner cavity of AuNCs and MPLA with its long hydrophobic alkyl chains was inserted into the liposomes. aCD11c was conjugated on the surface of Lipos-AuNCs to promote the activation and maturation of DCs, and subsequently enhance tumor specific T cell responses. Moreover, by using fluorescence and photoacoustic imaging, AuNCs accumulation and AuNCs-engulfed DCs migration to regional lymph nodes could be realtime visualized. Carbon nanotubes (CNTs). CNTs consisting of single or multiple layers of graphene with carbon atoms bonded in hexagonal lattices have been used in a wide range of medical applications.183 High surface area to volume ratio confers to CNTs the capacity of multiple attachment sites with proteins. Moreover, they are not immunogenic and the functionalized CNTs have the ability to cross biological membranes, deliver biomolecules into the cytoplasm, and protect the attached molecules against enzymatic degradation. Zakaria et al synthesized single-walled carbon nanotubes (SWCNTs) functionalized with TRAIL via noncovalent 1-pyrenebutanoic acid N-hydrosuccinimid ester (PSE) to mimic membrane-bound TRAIL, because membrane-bound TRAIL induces stronger receptor aggregation and apoptosis than soluble TRAIL.27 Batista de Faria et al developed an anticancer vaccine comprising multi-walled carbon nanotubes (MWCNTs) for the delivery of cancer testis antigen, NY-ESO-1, and Toll-like receptor agonist, CpG.93 They found that the vaccination induced strong CD4+ T as well as CD8+ T cell-mediated immune responses, significantly delaying the tumor development and prolonging the mice survival. Graphene oxide (GO) nanosheet. Graphene is comprised of a single layer of carbon atoms attached to each other by strong sp2 hybridized carbon-carbon bond.184 After oxidization, the oxidized portion on the surface of GO offers functional groups for protein conjugation and the un-modified region is capable of loading protein drugs via hydrophobic interactions and π-π stacking.13 Jiang and co-workers reported a graphene-based nanocarrier for the co-delivery of TRAIL and DOX.30 DOX was absorbed onto GO via supramolecular π-π stacking interaction, and a hetero-bifunctional PEG containing amino and azide terminal groups was used as linker to connect GO and furin-cleavable peptide, which could be specifically recognized and cleaved by furin. Finally, TRAIL was conjugated to the sulfhydryl of peptide. On the cancer cell membrane, 24


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furin digested the peptide linker, leading to the extracellular release of TRAIL and preventing its internalization into cell. Subsequently, DOX-bearing GO nanosheet was internalized into the cell to mediate DNA damage-mediated cytotoxicity. Magnetic nanoparticles. Magnetic nanoparticles have controllable physical and chemical properties, and have been extensively applied in theranostic studies and drug delivery.185 Particularly, the magnetic guidance can enhance the accumulation of cargoes in the tumor site. The most prominent magnetic nanoparticles are Fe3O4 and γ-Fe2O3 nanoparticles.186 Duan et al reported a multi-gradient targeting magnetic microbubble for cancer theranostics.28 Firstly, γ-Fe2O3 nanoparticles were chemically conjugated on the surface of polymer microbubbles. Then, arginine-glycine-aspartic acid-L-TRAIL (RGD-L-TRAIL) was precisely conjugated with magnetic microbubble to construct the molecularly targeted magnetic nanoparticles (MMBs). The MMBs could specifically accumulate in the tumor due to multigradient cascade targeting. After accurate diagnosis of tumor by magnetic resonance imaging (MRI), TRAIL effectively induced tumor cell apoptosis. Peptide/protein-based nanocarriers Cell-penetrating peptides (CPPs) are oligopeptides with potent membrane activities, which can deliver a wide range of membrane-impermeable bioactive agents across cell membranes.187 As such, they are intensively utilized to facilitate the intracellular delivery of protein/peptide cargoes that are originally impermeable to cell membranes. Additionally, majority of the CPPs are cationic peptides containing lysine and arginine residues, which could bind to negatively charged proteins to form nanocomplexes, which further enhance the cellular internalization as well as endolysosomal escape efficiency. Wang et al designed a synthetic CPP, Cys-Trp-Trp-Arg8-Cys-Arg8-Cys-Arg8-Cys, which formed nanocomposites with OVA via electrostatic self-assembly.84 The oxidization of thiol groups on cysteine residues induced interpeptide crosslinking to construct denser peptide/OVA condensates. The cell-penetrating peptides increased antigen uptake by APCs, and the antigens were rapidly released in the cytoplasm due to degradation of the disulfide bonds by intracellular glutathione. The strong membrane activity and high positive charge density of CPPs are also associated with potential systemic toxicity. To address this issue, Gao et al developed the electronically neutral cyclic peptide cyclosporine A (CsA) to deliver a membrane-impenetrable pro-apoptotic peptide (PAD).102 The CsA-PAD conjugate could achieve similar anti-tumor efficacy to TAT-PAD (PAD conjugated with TAT, a classical CPP) but with much lower heart and liver toxicity. Proteins or self-assembled peptides can also serve as nanocarriers to deliver protein drugs.8, 188 For example, bovine serum albumin (BSA) and human serum albumin (HSA) with small sizes and good biocompatibility have been widely used for drug delivery. Recently, Hu et al reported a charge designable and tunable green fluorescent protein (GFP)-base protein delivery system, His29GFP-RNase A. Because of the presence of histidines, the protein carrier is neutral at pH 7.2 while transforms to cationic at pH 6.5. As such, it can selectively permeate the cell membrane at pH 6.5 to efficiently escape from endosomes.61 RNase A could be released in the cytosol to cause substantial mRNA degradation in HeLa cells, and His29GFP-RNase A exhibited potent anti-tumor efficacy in various cell lines and 3D tumor spheroids at pH 6.5. DNA nanostructures The unique programmability of DNA has enabled the design of uniform nanostructure with 25



well-defined shape and hybridization for protein delivery. The superior biocompatibility was attributed to the degradability of DNA by DNase in tissues. Meanwhile, though free DNA is cell membrane-impermeable, DNA nanostructure could readily enter into the cells via endocytosis.189 Proteins could be loaded into DNA nanostructure through covalent conjugation, inter-molecular hybridization, and intra-molecular interaction.190 Moreover, the intrinsic multivalency confers the functionalization of DNA nanostructure.191 Recently, Kim et al developed a streptavidin-mirror DNA tetrahedron hybrid with a streptavidin providing a stoichiometrically controlled loading site for the enzyme and an L-DNA (mirror DNA) tetrahedron enabling the intracellular delivery potential for various enzymes.47 Biotinylated enzymes loaded on the streptavidin via specific affinity could be efficiently delivered into tumor cells. Zhang et al developed a DNA-affibody nanoparticle for inhibiting HER2-overexpressed breast cancer cells.192 On one hand, DNA tetrahedron was used to anchor two affibody molecules, which mimic antibody to specifically target HER2 receptors. On the other hand, DNA tetrahedron as a vehicle could non-covalently bind ~53 molecules of DOX. In breast cancer cells overexpressing HER2, DNA-affibody nanoparticles exhibit greater tumor cell inhibition than DOX. Conclusion and perspective Protein therapeutics revolutionizes the treatment of cancer by featuring high pharmacological efficiency and selectivity. Tremendous progresses have been made in protein delivery, and various systems/vehicles have been developed to protect cargo proteins against detrimental physiological environments and efficiently deliver proteins to tumor sites and cells. Despite these remarkable achievements, further development is necessary to translate these fundamental researches into preclinical or clinical investigation and finally enter the market. Multiple challenges with respect to stability, biocompatibility, targeting efficiency, systemic toxicity, and immunogenicity still need to be properly addressed. Proteins/peptides are structurally different from nucleic acids that contain similar repeating units, and different proteins usually have different MWs, charge (either positive or negative), hydrophobicity, and higher-ordered structure (α-helix or β-sheet). Thus, a protein carrier is usually suitable for the encapsulation of particular proteins/peptides. With regard to this, a universal yet potent protein delivery system is highly demanded. In terms of existing protein encapsulation/conjugation strategies, covalent modification may cause irreversible denaturation, and low yield of the conjugation chemistry would hurdle its translation by greatly increasing the cost. Physical adsorption may lead to premature cargo release. For intracellular delivery, most of the nanocarriers still lack sufficient potency in terms of transmembrane delivery, leading to undesired cellular internalization and severe endolysosomal entrapment. While utilization of CPPs could overcome the membrane barrier, their poor cell selectivity along with undesired toxicity raises another critical concern. Design of intelligent vehicles can render multi-functions to overcome the afore-mentioned barriers, while it will inevitably increase the synthesis/formulation complexity and the cost, leading to difficulty in quality control and mass production. Additionally, the biocompatibility, biodegradability, and potential toxicity of degraded bi-produces need to be seriously considered. For an ideal formulation, protein cargoes should be efficiently loaded and be protected against degradation before arriving at targeting sites. At the functional sites, protein cargoes should be rapidly released and restore their activity. Formulations with simple but effective architectural design are highly necessitated to boost the chance for clinical translation. 26


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (L. Yin); [email protected] (Y. Ji) ORCID Cyrille Sabot: 0000-0002-4573-0555 (L. Yin) Author Contributions Xun Liu and Fan Wu contributed equally. Notes The authors declare no competing financial interest. Acknowledgements This work was supported by the Ministry of Science and Technology of China (2016YFA0201200), the National Natural Science Foundation of China (51722305, 51573123, and 51873142), and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References (1) Gu, Z., Biswas, A., Zhao, M., and Tang, Y. (2011) Tailoring nanocarriers for intracellular protein delivery. Chem. Soc. Rev. 40, 3638-3655. (2) Mitragotri, S., Burke, P. A., and Langer, R. (2014) Overcoming the challenges in administering biopharmaceuticals: Formulation and delivery strategies. Nat. Rev. Drug Discovery 13, 655-672. (3) Mocellin, S., Rossi, C. R., Pilati, P., and Nitti, D. (2005) Tumor necrosis factor, cancer and anticancer therapy. Cytokine Growth Factor Rev. 16, 35-53. (4) Scott, A. M., Wolchok, J. D., and Old, L. J. (2012) Antibody therapy of cancer. Nat. Rev. Cancer 12, 278-287. (5) He, C., Tang, Z., Tian, H., and Chen, X. (2016) Co-delivery of chemotherapeutics and proteins for synergistic therapy. Adv. Drug Delivery Rev. 98, 64-76. (6) Leader, B., Baca, Q. J., and Golan, D. E. (2008) Protein therapeutics: A summary and pharmacological classification. Nat. Rev. Drug Discovery 7, 21-39. (7) Cicero, A. F. G., Fogacci, F., and Colletti, A. (2017) Potential role of bioactive peptides in prevention and treatment of chronic diseases: A narrative review. Br. J. Pharmacol. 174, 1378-1394. (8) Qi, G.-B., Gao, Y.-J., Wang, L., and Wang, H. (2018) Self-Assembled Peptide-Based Nanomaterials for Biomedical Imaging and Therapy. Adv. Mater. 30, 1703444. (9) Postupalenko, V., Desplancq, D., Orlov, I., Arntz, Y., Spehner, D., Mely, Y., Klaholz, B. P., Schultz, P., Weiss, E., and Zuber, G. (2015) Protein Delivery System Containing a Nickel-Immobilized Polymer for Multimerization of Affinity-Purified His-Tagged Proteins Enhances Cytosolic Transfer. Angew. Chem. Int. Ed. 54, 10583-10586. (10) Zuris, J. A., Thompson, D. B., Shu, Y., Guilinger, J. P., Bessen, J. L., Hu, J. H., Maeder, M. L., Joung, J. K., Chen, Z.-Y., and Liu, D. R. (2015) Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73-80. (11) Nischan, N., Herce, H. D., Natale, F., Bohlke, N., Budisa, N., Cardoso, M. C., and Hackenberger, C. P. R. (2015) Covalent attachment of cyclic TAT peptides to GFP results in protein delivery into live cells with immediate bioavailability. Angew. Chem. Int. Ed. 54, 1950-1953. (12) Brodin, J. D., Sprangers, A. J., McMillan, J. R., and Mirkin, C. A. (2015) DNA-Mediated cellular delivery of functional enzymes. J. Am. Chem. Soc. 137, 14838-14841. 27



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Peptide Delivery

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