Small Delivery Vehicles of siRNA for Enhanced Cancer Targeting

Subscriber access provided by Kaohsiung Medical University

Review

Small Delivery Vehicles of siRNA for Enhanced Cancer Targeting Hyun Jin Kim, Yu Yi, Ahram Kim, and Kanjiro Miyata Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00546 • Publication Date (Web): 04 Jun 2018 Downloaded from http://pubs.acs.org on June 5, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Small Delivery Vehicles of siRNA for Enhanced Cancer Targeting

1 2

Hyun Jin Kim1, Yu Yi2,3, Ahram Kim4, Kanjiro Miyata2,*

3 4 5

1

6

University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

7

2

8

Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

9

3

Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The

Department of Materials Engineering, Graduate School of Engineering, The University of

CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biological Effects of

10

Nanomaterials and Nanosafety, National Center for Nanosciecne and Technology, No. 11

11

Beiyitiao, Zhongguancun, Beijing 100190, China

12

4

13

Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8573, Japan

Department of Materials Science, Graduate School of Pure and Applied Sciences, University of

14 15

*

16

K. Miyata: [email protected]

Corresponding Authors:

17 18

Abstract

19

Small interfering RNA (siRNA) drugs have been considered to treat various diseases in major

20

organs. However, siRNA drugs developed for cancer therapy are hindered from proceeding to

21

clinics. To date, various delivery formulations have been developed from cationic lipids,

22

polymers, and/or inorganic nanoparticles for systemic siRNA delivery to solid tumors. Most of

23

these delivery vehicles do not generate small particle sizes and pharmacokinetics required for

24

accumulation in target cancer cells, compared with clinically tested anticancer drug-loaded

25

polymeric micelles. This review describes the significance of small, long-circulating vehicles for 1 ACS Paragon Plus Environment

Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

efficient delivery of siRNA to cancer tissues via the enhanced permeability and retention (EPR)

2

effect. We summarize recent biological evidence that supports the size effect of delivery vehicles

3

in tumor microenvironments and introduce the promising strategies for construction of small

4

vehicles with a size of 10 to 50 nm. We then discuss the feasibility of these delivery vehicles

5

with respect to translation to clinical trials.

6 7

Keywords: RNAi, siRNA delivery, cationic block copolymer, cancer targeting, EPR effect.

8 9

1. Introduction

10

Small interfering RNA (siRNA) drugs have been gradually developed to treat various

11

intractable diseases and have been tested in clinical trials. A post-transcriptional gene silencing

12

mechanism, termed RNA interference (RNAi), is triggered by siRNA in mammalian cells.1 As a

13

double-stranded RNA molecule, siRNA typically has 21–23 nucleotides in each strand. In

14

cytoplasm, siRNA binds with a protein complex, called the RNA-induced silencing complex

15

(RISC). The sense strand of the siRNA is cleaved and ejected.2,3 The antisense strand

16

thermodynamically selects complementary messenger RNA (mRNA). Antisense strands with

17

partial target hybridization promote translational repression, and those with perfect

18

complementary hybridization induce mRNA degradation by cleavage of the phosphodiester bond

19

at the mRNA position paired with antisense strand nucleotides 10 and 11.3 Drugs based on

20

siRNA have potential advantages over traditional drugs in cancer therapy. RNAi can inhibit the

21

targets, which are inaccessible to traditional drugs, by using mRNA genetic information.4 KRAS

22

protein was dismissed as “undruggable” for many years until recent research showed that

23

covalent inhibitors bind and selectively inhibit one of the KRAS mutant isoforms (KRAS

24

G12C).5,6 This was because the KRAS protein did not appear to present suitable pockets, except

25

for the GDP/GTP-binding site. The KRAS protein binds very tightly to these nucleotides, which

26

makes identifying competitive nucleotide analogs virtually impossible.7 Drugs based on siRNA

27

provide an alternative approach to controlling KRAS expression and have proven efficacious in

28

animal models of various tumors.8,9 Furthermore, RNAi is suitable for gene silencing in multiple

29

target mRNAs, which induces therapeutic synergy. Certain malignant tumors depend on a 2 ACS Paragon Plus Environment

Page 2 of 41

Page 3 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

developmental hierarchy, in which many transcription factors (TFs) can function as oncogenes.10

2

Glioblastoma (GBM) stem-like cells drive tumor propagation and therapeutic resistance. Thus,

3

TFs in GBM stem-like cells may function as novel therapeutic targets. One study demonstrated

4

that simultaneous RNAi on four TFs (SOX2, OLIG2, SALL2, and POU3F2) successfully

5

suppressed the growth of brain tumor-initiating cells in a murine brain tumor model.11

6

For RNAi in the body, siRNA must be transported into target cells after administration.

7

Native (or unmodified) siRNA molecules are degraded by nucleases in blood and hydrolyzed in

8

lysosomes. Thus, siRNA molecules have been chemically modified or formulated into delivery

9

vehicles to enhance their availability. Chemical modification of internucleotide linkages and

10

oligonucleotide sugars enhances nuclease resistance and decreases the immunostimulatory

11

properties of native siRNA.12 Various synthetic materials, such as cationic polymers, lipids (or

12

lipidoids), and inorganic materials, have been employed to formulate multimolecular assemblies

13

(i.e., delivery vehicles) with siRNA.13‒15 These have been improved to increase the transport

14

efficacy of therapeutic siRNA to achieve more efficient RNAi in target cells. The vehicles

15

protect vulnerable siRNA from enzymatic degradation and prevent rapid renal filtration and non-

16

specific interaction with serum proteins after systemic administration. Once the delivery vehicles

17

reach target cells, they (i) facilitate internalization in the target cells through adsorptive (or

18

receptor-mediated) endocytosis, (ii) escape from the endosome to the cytoplasm, and (iii) finally

19

release the siRNA to be loaded into the RISC [Fig. 1]. For cancer therapy, the vehicles need to

20

reach into cancer cell nests while avoiding accumulation in major organs. Tumor vessels lack

21

tight endothelial monolayers and thus the vehicles can pass through (penetrate) these vasculature

22

defects (e.g., pores).16 This phenomenon is known as the enhanced permeability and retention

23

(EPR) effect.17 The penetration behavior of vehicles is mainly governed by diffusion with the

24

assumption that their sizes are smaller than those of the vascular defects. Thus, the vehicle size

25

(or diffusion coefficient) and concentration gradient between bloodstream and tumor tissue are

26

the main factors affecting tumor accumulation profiles (see the section 2.2 in this review).

27

Higher concentration gradients are obtained by longer circulation of vehicles in the bloodstream

28

as well as higher dosing. These facts emphasize the significance of delivery vehicles with

29

smaller size and longer circulation property in achieving more efficient cancer targeting based on

30

the EPR effect. This review (i) describes the recent biological evidence supporting the 3 ACS Paragon Plus Environment


1

significance of small vehicles; (ii) summarizes promising fabrication techniques of the small

2

vehicles for siRNA delivery; and (iii) discusses the feasibility of these delivery vehicles for

3

translation to clinical trials.

4

5 6

Figure 1. Schematic illustration of siRNA delivery in stroma-rich tumor microenvironments. (1)

7

Long-circulating delivery vehicles maintain a concentration gradient between bloodstream and

8

tumor tissue, facilitating their diffusion into the tumor tissue. (2) Small vehicles easily penetrate

9

vascular pores with heterogeneous sizes in tumor lesions. (3) Small vehicles easily diffuse into

10

stroma region and reach cancer cell nests. (4) Vehicles are endocytosed into cancer cells, escape

11

from late endosomes/lysosomes to the cytoplasm, and release siRNA payloads. Finally, the

12

siRNA molecules are loaded into RISC and induce RNAi.

13

4 ACS Paragon Plus Environment

Page 4 of 41

Page 5 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Biomacromolecules

2. Behavior of delivery vehicles in tumor microenvironments

2

Molecules in the bloodstream are transported into tissue area by different routes. Gases

3

(O2 and CO2), lipophilic solutes, and small hydrophilic solutes freely pass (or diffuse) through

4

capillary endothelia.18,19 In continuous capillary blood vessels, macromolecules pass through

5

endothelia by transcellular and/or paracellular routes. The transcellular route requires active

6

transport mechanisms using caveolae and vesiculo-vacuolar organelles. The paracellular route is

7

mediated by the coordinated opening and closing of the intercellular junctions of endothelial

8

cells (e.g., vascular pores).18,19 Macromolecules larger than ~40 kDa in molecular weight (MW)

9

cannot spontaneously extravasate through these continuous capillary vascular pores.20 In tumor

10

vessels, vascular endothelial growth factor (VEGF), which is secreted by cancer cells, weakens

11

intercellular junction and induces the increase of paracellular permeability.21 Consequently,

12

macromolecules larger than ~40 kDa can extravasate from this leaky vasculature to the tumor

13

mass when their sizes are smaller than those of tumor vascular pores, which vary even in the

14

same tumor tissue (see the section 2.1 in this review). Smaller macromolecules more rapidly

15

extravasate than larger ones, following two reasons. One is that smaller macromolecules have

16

more opportunities for extravasation by penetrating even smaller vessel pores. The other is that

17

the penetration behavior of macromolecules is mainly governed by diffusion.22 Dextran with a

18

MW of 3.3 kDa (~3 nm in hydrodynamic diameter (DH)) penetrated two orders of magnitude

19

faster than dextran with a MW of 2 MDa (~50 nm in DH) in a tumor tissue in human squamous

20

tumor-bearing mice, as estimated by the Kedem–Katchalsky equation.23 Intravenously

21

administered delivery vehicles extravasate from the bloodstream and reach cancer cell nests via

22

three transport steps [Fig. 1]. Firstly, the vehicles penetrate undeveloped and leaky vasculature

23

and reach a tumor tissue. When cancer cells surround the blood vessels, the vehicles immediately

24

reach the cancer cells. In stroma-rich tumors, the blood vessels are frequently surrounded with

25

extracellular matrices. In this case, the vehicles need to go through the matrices while avoiding

26

non-specific absorption to collagens and sulfated glycosaminoglycans. Then, the vehicles reach

27

the cancer cell nests and are internalized by target cells.

28

2.1. Significance of size of delivery vehicles. Tumor accumulation of macromolecules by the

29

EPR effect remains controversial because of disagreement regarding the vascular pore cutoff size

30

in solid tumors (e.g., human cancer patients vs. experimental animal models). Early studies on 5 ACS Paragon Plus Environment


1

the vascular pore cutoff size reported extremely leaky tumor blood vessels in murine ectopic

2

models, with pore sizes between 380 nm and 780 nm [Fig. 2A-C].24,25 However, recent studies

3

suggested that a realistic pore size in patients should be less than a few hundred nanometers.26

4

Heterogeneous tumor accumulation by the EPR effect is observed in the same tumor tissue or

5

individual patient. Cancer patients develop metastases in different organs. Tumors at the

6

metastatic sites are exposed to different cellular compositions and extracellular matrices, leading

7

to heterogeneous EPR effects. In 19 human patients with HER2-positive metastatic breast cancer,

8

a HER2-targeted poly(ethylene glycol)-conjugated (PEGylated) liposome (~100 nm in DH)

9

exhibited a 35 fold higher accumulation from 24 to 48 h (0.52–18.5% ID/kg) in bone and brain

10

tumor lesions.26 The accumulation amounts in an individual patient (patient 02) exhibited a 20-

11

fold difference between the highest (10% ID/kg) and the lowest (0.5% ID/kg) values in two

12

different tumor lesions [Fig. 3]. Metastatic tumor lesions in various organs exhibited different

13

tumor accumulation profiles. Among all the metastatic tumors in 19 patients, tumor lesions in the

14

liver generally exhibited higher tumor accumulation than those in other organs (e.g., breast, bone,

15

lymph node, lung, skin, mediastinum, and chest wall). Another study observed the heterogeneity

16

of the EPR effect in canine tumors. In 11 canines with spontaneous solid tumors, 110 nm-sized

17

PEGylated liposomes exhibited widely varying tumor accumulation profiles.27 In detail, the

18

liposomes displayed high accumulations in six of seven carcinomas, whereas the same liposomes

19

accumulated in only one of four sarcomas. Biological differences between tumors were not

20

clearly explained in the above two studies. It is probable that such tumor lesions in patients have

21

different vascular pore sizes. In achieving accumulation in various tumor lesions in all organs,

22

downsizing of delivery vehicles will be one of the most plausible approaches to maximizing the

23

EPR effect in heterogeneous tumors. Size-dependent tumor accumulation of vehicles was clearly

24

observed in lymph node metastases in a murine model.28 A 30 nm polymeric micelle

25

significantly accumulated in lymph node metastases, whereas an 80 nm PEGylated liposome

26

could barely penetrate the metastatic masses [Fig. 4]. Similarly, 18 nm lipid-coated calcium

27

phosphate nanoparticles (LCP-NPs) also exhibited more efficient accumulation in murine models

28

of various subcutaneous or orthotopic tumors, compared to 65 nm LCP-NPs.29 Note that these

29

two LCP-NPs had similar blood circulation properties (half-lives = ~4 h).

30


Page 6 of 41

Page 7 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1 2

Figure 2. (A) Scanning electron microscopic (SEM) image of luminal surface of normal blood

3

vessel in mouse mammary gland. The surface is smooth and has tight endothelial junctions. (B)

4

SEM image of tumor blood vessel in MCa-IV mouse mammary carcinoma. The vessel has

5

widened intercellular spaces, overlapping endothelial cells. (C) SEM image of high

6

magnification of a hole (arrows) in the endothelium in MCa-IV mouse mammary carcinoma.

7

Arrowheads indicate basement membrane filaments. Scale bar in (C) applies to all panels in 5

8

µm in (A); 2 µm in (B); 0.5 µm in (C). Reprinted by permission from Springer Nature, ref [24],

9

Copyright 2003. https://www.nature.com/articles/nm0603-713

10

11 64

12

Figure 3. Tumor deposition of doxorubicin-loaded

Cu-labeled HER2-targeted PEGylated

13

liposome (64Cu-MM-302) in individual patients with metastatic breast cancer was shown to be

14

highly variable. Each patient with multiple lesions was measured by positron emission



1

tomographic scans. Reprinted from [26]. Copyright 2017, with permission from American

2

Association for Cancer Research.

3

4 5

Figure 4. Intravital microscopic analyses of metastatic lymph nodes after co-injection of 30 nm

6

(1,2-diaminocyclohexane)platinum(II) (DACHPt)-loaded polymeric micelles (green) and 80 nm

7

PEGylated liposomes, Doxil (red). Quantification of the changes in the fluorescence signals

8

within the metastatic region (enclosed area by dotted line). Reprinted with permission from [28].

9

Copyright 2015, American Chemical Society.

10 11

In cancer cell nests (or tumor mass), delivery vehicles need to reach more cancer cells. It was

12

suggested that poorly developed intratumoral lymphatic vessels are functionally compromised,30

13

resulting in interstitial hypertension. This elevated interstitial fluid pressure compromises

14

convective transport of vehicles.22 Thus, the dominant mechanism of vehicle transport within

15

tumors should be diffusion. The diffusion coefficient of biologically inert (or stealth) vehicles

16

depends on the particle sizes and the constituents of tumor matrices. Smaller nanoparticles

17

exhibited longer penetration distances in tumor mass. A 25 nm polymeric micelle diffused from

18

the blood vessels to an average distance of 42 ± 7 µm in a murine model of a subcutaneous

19

breast MDA-MB-468 tumor, whereas a 60 nm polymeric micelle showed an average distance of

20

23 ± 4 µm.31 The similar results were also observed for PEGylated 12 nm quantum dots,32 20 nm

21

gold nanoparticles,33 and 30 nm polymeric micelles.34 These nanoparticles diffused more deeply

22

from tumoral blood vessels, compared with control nanoparticles with sizes of larger than 50 nm. 8 ACS Paragon Plus Environment

Page 8 of 41

Page 9 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

In fibrotic tumors, delivery vehicles also need to penetrate the stroma (or fibroblast- and

2

collagen-rich) tissue to reach the cancer cell nests. In clinical tumors, e.g., breast, pancreatic,

3

colorectal, and non-small-cell lung cancers, stromal tissues surrounding the blood vessels are

4

observed [Fig. 5A].35 These stromal tissues obstruct the diffusion of delivery vehicles and act as

5

a physical barrier to vehicle penetration. Indeed, subcutaneous BxPC3 tumor tissues are

6

equipped with fibrotic tissues that surround the blood vessels.36,37 Two different sized polymeric

7

micelles were separately prepared with DH of 30 nm and 70 nm and were systemically

8

administered into the same BxPC3 tumor-bearing mice.38 The 30 nm micelles were uniformly

9

distributed in the cancer cell nest. In contrast, the 70 nm micelles could not enter it. Although

10

some portions of 70 nm micelles penetrated the tumor vessel, they were entrapped close to a

11

blood vessel [Fig. 5B]. Consequently, the 30 nm micelles efficiently reduced the growth rate of

12

the pancreatic tumor model, whereas the 70 nm micelles were ineffective. This research clearly

13

demonstrates that a smaller vehicle is essential for increasing the delivery efficacy in stroma-rich

14

tumors. Meanwhile, it is reported that diffusion of vehicles in tumor tissues depends on the

15

collagen content.22 When virus-derived nanoparticles with a size of 150 nm were intratumorally

16

administrated into human melanoma tumor grown in a dorsal skinfold chamber, collagen regions

17

with higher density displayed a lower concentration of the nanoparticles compared with those

18

with lower density.39 These results suggest that collagen contents might be the major determinant

19

for vehicle diffusion in stroma tissues. Similarly, the penetration of 35 nm LCP-NPs into cancer

20

cell nests was greatly enhanced in the stroma-rich bladder cancer xenograft model after the

21

treatment of Quercetin, which is a small molecule used to decrease both fibroblast population

22

and collagen content.40

23



1 2

Figure 5. (A) Patient tumor stromal architecture. A tumor structure dominated by a pattern of

3

cancer cell nests surrounded by well-developed stromal structures, which contain the majority of

4

the vessels. Tumor cells were stained with DAPI (blue). Myofibroblast-like cells and blood

5

vessels were stained by α-smooth muscle actin (red) and CD31 (green), respectively. Reprinted

6

from [35]. Copyright 2013, with permission from American Association for Cancer Research.

7

(B) Z-stack volume reconstruction of BxPC3 tumors 1 h after co-injection of fluorescently

8

labeled 30 nm (green) and 70 nm (red) DACHPt-loaded polymeric micelles. Reprinted by

9

permission from Springer Nature, ref [38]. Copyright 2011.

10

https://www.nature.com/articles/nnano.2011.166

11 12

2.2. Significance of blood circulation properties of delivery vehicles. In the bloodstream,

13

vehicle transport in the flow direction is dominated by convection. Transfer of vehicles from the

14

bloodstream to tumor tissues is dominated by concentration-dependent diffusion because the

15

flow velocity in the radial direction is much slower than that in the flow direction.41 Additionally,

16

the interstitial fluid pressure elevated in tumor tissues reduces the pressure gradient across the

17

vessel wall, which further compromises the convection from the blood to the tumor tissues.22 The

18

concentration gradient of vehicles between the bloodstream and tumor tissues is maintained in a

19

certain extent if they can steadily circulate in the bloodstream. Based on the concentration


Page 10 of 41

Page 11 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

gradient, vehicles with sizes of several tens nanometer slowly diffuse into tumor tissue during

2

circulation. Thus, long-circulating vehicles are advantageous for enhanced tumor accumulation.

3

Herein, the blood circulation property of delivery vehicles is discussed based on the

4

pharmacokinetics. Classic pharmacokinetics describes that drug elimination rate follows the

5

first-order reaction, where the drug concentration exponentially decreases with time.42 Also,

6

pharmacokinetic studies on drug delivery have utilized a two-compartment model, where the

7

drug elimination is represented by two different phases: first distribution (or α) phase and second

8

elimination (or β) phase. The distribution phase is generated by the drug distribution into

9

peripheral compartment, which is significantly slower than the instantaneous drug distribution

10

into central compartment after administration [Fig. 6A]. Accordingly, a slope in the distribution

11

phase is affected by the drug elimination and the drug distribution/redistribution. Once the

12

distribution and redistribution reach equilibrium, the distribution phase shifts to the elimination

13

phase featuring a less negative slope. Small drugs are instantaneously distributed into highly

14

perfused organs, e.g., heart, lungs, kidneys, liver, and brain, as well as blood, whereas not into

15

less well-perfused tissues, e.g., muscle, fat, and skin. Thus, the highly perfused organs and the

16

less well-perfused tissues are considered as central compartments and peripheral compartments,

17

respectively. In contrast, macromolecular drugs generally display substantially slow distribution

18

even in the highly perfused organs. This indicates that blood and organs/tissues should

19

correspond to central compartments and peripheral compartments, respectively, in the

20

macromolecular drug delivery. In this regard, stable delivery vehicles follow a two-compartment

21

model with a fairly long α-phase or apparently single-compartment model without distribution

22

phase [Fig. 6B].42 The gentle slope in the profile of polymeric micelles indicates that they stably

23

circulated in the bloodstream without rapid organ/tissue distribution. This profile is in sharp

24

contrast with that of small drugs (oxaliplatin), which disappeared from the blood much faster in a

25

clear two phase-manner.

26

A confocal laser scanning microscopic (CLSM) technique enables direct and real-time

27

observation of fluorescent nanoparticles (or macromolecules) circulating in the bloodstream.43

28

The obtained time profile of fluorescence intensity represents the circulation property, including

29

stability, of nanoparticles in the blood [Fig. 6C]. A rapid decline in the initial fluorescence

30

intensity indicates that the nanoparticles were rapidly eliminated from the bloodstream, possibly 11 ACS Paragon Plus Environment


1

due to distribution into organs/tissues following nanoparticle dissociation. It should be noted that

2

the PEGylated liposome on the market and the polymeric micelles tested in clinical trials show

3

half-lives from ten to hundred hours in the blood circulation, as summarized in Table 1. A

4

HER2-targeted PEGylated liposome (half-life = 33 h in patients) continuously accumulated in a

5

tumor for 72 h and achieved high tumor accumulation (2.3–10.6% ID/kg) in some tumor lesions,

6

as described above.26 A cisplatin-loaded polymeric micelle, which completed phase II clinical

7

trials44 and is being evaluated in phase III clinical trials, exhibited half-lives of 129 ± 40 h in a

8

human patient45 and approximately 8 h in a mouse.46 This micelle (DH = 25 ± 1 nm) continuously

9

accumulated in a murine model of a subcutaneous pancreatic BxPC3 tumor for 24 h and

10

achieved approximately 5% ID/g.47 These data suggest that siRNA delivery vehicles also need

11

longer blood circulation property for efficient tumor accumulation. An siRNA-loaded lipid

12

nanoparticle (LNP) tested in clinical trials is reported to have half-lives of 1–2 h in patients

13

[Table 2], which are considerably shorter than those of PEGylated liposomes and cisplatin-

14

loaded polymeric micelles. The fabrication of long-circulating vehicles for siRNA delivery is

15

technically more difficult compared with low MW anticancer drugs, because of the rigid

16

cylindrical architecture (2 × 6 nm in size) of siRNA with high hydrophilicity.

17

Several previous studies reported the relationship between nanoparticle size and blood

18

circulation property. The 30 and 50 nm polymeric micelles prepared from PEG-b-poly(amino

19

acid)s showed no significant differences in blood circulation profiles (half-lives = 14 h for both

20

polymeric micelles).38 Also, 18 and 65 nm lipid-coated calcium phosphate nanoparticles showed

21

the similar blood circulation profiles with half-lives of 4 h.29 However, the effect of nanoparticle

22

size of less than 50 nm on the blood circulation property remains to be investigated.

23


Page 12 of 41

Page 13 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

2 3

Figure 6. (A) A two-compartment model for drug elimination. (B) Time profiles of platinum

4

concentration in the plasma after intravenous administration of DACHPt-loaded polymeric

5

micelles (■) and free oxaliplatin (◊), determined by ion coupled plasma-mass spectrometry.

6

Reprinted from [48]. Copyright 2004, with permission from Elsevier. The polymeric micelle

7

exhibits a long α-phase with a half-life of > 9 h. In contrast, a small molecule, oxaliplatin, is

8

cleared rapidly from circulation. (C) A representative blood circulation profile of fluorescent-

9

labeled antisense oligonucleotide-loaded polymeric micelles, determined by intravital real-time

10

CLSM observation. The polymeric micelles were intentionally prepared to show a short blood

11

circulation profile with a half-life of ~8 min in α phase. Unpublished data.

12

Table 1. Representative long-circulating delivery vehicles for cancer therapy.13,49

13 Delivery

Name

Drug

Size

vehicle Polymeric

NK012

SN-38

20

NC-6004

137 ± 19 h in

II

Triple negative

cisplatin

25

129 ± 40 h in

breast cancer III

Pancreatic cancer

I

Various solid

patient NC-4016

micelle PEGylated

Disease

patient

micelle Polymeric

Phase

(nm)

micelle Polymeric

Half-life

(1,2-diaminocyclohexane)

30

10–50 h in mice

platinum(II) (DACHPt) Doxil

doxorubicin

tumors 90

liposome

21–90 h in

On the

patient

market

14


Various tumors


1

3. siRNA-based drugs tested in clinical trials for cancer therapy

2

This section introduces current siRNA delivery vehicles tested in clinical trials [Table 2].

3

Major advances have been achieved for liver diseases, including transthyretin (TTR)-mediated

4

amyloidosis, hypercholesterolemia, and hepatitis B virus infection, because of the histological

5

characteristics. Sinusoidal capillaries in liver have larger intercellular pores (~150 nm),

6

compared with continuous capillary blood vessels, and a discontinuous basement membrane,

7

allowing the easy access of nanoparticles to hepatocytes.50,51 The presence of various receptors

8

(e.g., asialoglycoprotein, retinol binding protein, and low-density lipoprotein receptor) on

9

hepatocytes can further facilitate the cellular uptake of delivery vehicles equipped with targeting

10

ligands.52–54 Importantly, the duration of RNAi-induced silencing effects is correlated to the

11

doubling time of target cells. Under cultured conditions, the RNAi activity continued for 3–7

12

days in rapidly dividing cells (e.g., doubling time ≈ 1 day) and for several weeks in non-dividing

13

cells.55 Dilution of intracellular siRNA associated with the cell division mainly affects the

14

duration of gene silencing. Thus, the siRNA-mediated gene silencing in the liver, where

15

hepatocytes are quiescent and rarely replicated in the normal status,56 can be sustained for long

16

periods.

17

Delivery vehicles targeting the liver have been extensively optimized with respect to the

18

endosome-escapability of cationic components57,58 or the targetability using ligands.52,53 Among

19

them, Patisiran (ALN-TTR02) has completed Phase III trials for the treatment of TTR-mediated

20

amyloidosis. Patisiran is an LNP formulation comprising a synthetic amino lipid (DLin-MC3-

21

DMA), distearoylphosphatidylcholine (DSPC), cholesterol, and PEG-lipid. The amino lipid,

22

DLin-MC3-DMA, was selected to have an appropriate ionizable property (or pKa = 6.44) from a

23

large pool of lipid candidates for enhanced endosomal escape.57 The LNP had a size distribution

24

from 70 nm to 90 nm and a polydispersity index of 0.11 ± 0.04.57 Two doses of Patisiran every 3

25

weeks in a Phase II study reduced the mean serum TTR level by approximately 80%, and the

26

RNAi effect continued for approximately 110 days.59

27

For siRNA delivery to cancer, two LNP formulations, Atu027 and ALN-VSP02, have

28

completed their Phase I/II and I trials, respectively. Atu027 is composed of four components, a

29

cationic lipid (AtuFECT01), a neutral helper lipid, a PEGylated lipid, and a 23-mer blunt-ended


Page 14 of 41

Page 15 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

siRNA for targeting protein kinase N3 (PKN3).60,61 Atu027 has an average DH of 120 nm with a

2

size distribution in the range of 30–480 nm.62 A pharmacokinetic analysis of siRNA and two

3

lipid components (AtuFECT01 and a neutral helper lipid) was carried out for 4 h of infusion.60

4

The antisense strand of siRNA has a half-life of only 1 h in plasma. Interestingly, the two lipid

5

components exhibited much longer half-lives in plasma of ~40 h. This result indicates that

6

siRNA was immediately detached from the surface of Atu027 in the bloodstream. ALN-VSP02

7

is

a

LNP

composed

of

five

components,

a

cationic 58

lipid

(DLinDMA),

8

distearylphosphatidylserine, cholesterol, PEG-lipid, and siRNA.

Two different siRNAs

9

targeting VEGF-A and kinesin spindle protein (KSP) are mixed at a 1:1 molar ratio.63 ALN-

10

VSP02 has a DH of 80 nm to 100 nm with a ζ-potential of +6 mV at pH 7.4. A pharmacokinetic

11

analysis of two different siRNAs after 15 min of infusion showed a rapid decline in the plasma

12

concentration (half-life = 1‒2 h).63 Biopsies from 12 patients were subjected to quantitative RT-

13

PCR to measure VEGF and KSP mRNA levels; most of the patients showed no difference in the

14

amounts of VEGF (8 patients) and KSP mRNA (10 patients) relative to the pretreatment biopsy.

15

In Atu027 and ALN-VSP02, the cationic lipid components (AtuFECT01 and DLinDMA) were

16

developed to provide LNPs with higher surface charges permitting the effective siRNA loading

17

and more efficient endosome-escapability, respectively. Nevertheless, the clinical results suggest

18

that these design parameters should be insufficient to elicit successful RNAi for cancer therapies.

19

The sizes and blood circulation properties of these vehicles should not be suitable for efficient

20

tumor accumulation.

21 22

Table 2. RNAi-based drug examples tested in clinical trials for liver diseases and cancer therapy. Delivery

Name

Target

Size (nm)

Half-life

Phase

Disease (organ)

ALN-PCSsc

PCSK9

6

5.8–11 h in

II

Hypercholesterolemia

vehicle GalNAcsiRNA

a,b52

LNP

57,59

LNP

53

c

(Inclisiran) ALN-TTR02

patient TTR

70–90

NP

d

(Liver) III

(Patisiran) ND-L02-s0201

Hereditary

ATTR

amyloidosis (Liver) Hsp47

153 ± 29

ND

e

I

Hepatic (Liver)


fibrosis


LNP60

Atu027

PKN3

120

~1

Page 16 of 41

h

in

I/II

patients LNP

63

LNP

64

ALN-VSP02

VEGF,

80–100

KSP siRNA-EphA2-

EphA2

1–2

h

Advanced

solid

tumor in

I

Liver cancer

I

Advanced tumor

patients ND

ND

DOPC

1

a: subcutaneous administration, b: the same delivery system is applied for other diseases. ALN-AS1 (Givosiran) for

2

acute hepatic porphyrias, ALN-AT3 (Fitusiran) for hemophilia and rare bleeding disorders, ALN-CC5 (Cemdisiran)

3

for complement-mediated diseases, ALN-GO1 (Lumasiran) for primary hyperoxaluria type 1, and ALN-TTRsc02

4

for hereditary ATTR amyloidosis. c: half-life in β phase, the data from the same delivery system, Revusiran,65 d: not

5

provided. Area-under-curve and Cmax values are provided. e: no data.

6 7

4. Construction of small delivery vehicles

8

This section introduces construction techniques of small delivery vehicle of siRNA with a

9

size of 10 to 50 nm. Although some examples described in this section were developed for

10

delivery of single-strand RNA or short DNA with the length of less than 30 bases, they should be

11

readily applied for the delivery of siRNA due to their structural similarity. Section 2 highlighted

12

the important roles of particle size for efficient blood extravasation and tumor penetration. Other

13

physicochemical factors (e.g., surface chemistry, charge, and shape) also affect the performances

14

of delivery vehicles, yet not explained in detail here because (i) most of small delivery vehicles

15

are equipped with PEG to reduce non-specific absorption of blood components, such as serum

16

proteins; (ii) the PEG layer provides nearly neutral surface charges to the vehicles; and (iii) most

17

of multimolecular assemblies in the rage of 10–50 nm are spherical. Other reviews summarize

18

the impact of surface chemistry,13 charge,66 and shape67,68 of delivery vehicles.

19 20

4.1. Polymer-conjugated siRNA delivery vehicles

21

One of the simplest siRNA delivery vehicles is a covalent conjugate of siRNA and

22

polymer, such as linear or branched PEGs. Polymer-siRNA conjugates can avoid kidney

23

filtration because of their larger size compared to the pore size (approximately 6‒10 nm) of the

24

glomerular basement membrane.69 Linear PEG chains with MWs of 5,600 and 13,400 are 16 ACS Paragon Plus Environment

Page 17 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

calculated to have DH values of 6 and 10 nm, respectively, according to the random coil

2

conformation model.70 Thus, PEG molecules with a MW above ~10,000 should be essential for

3

enhancing the blood circulation properties of polymer-siRNA conjugates. Indeed, an

4

intravenously administered PEG-aptamer conjugate, containing a PEG molecule with a MW of

5

40,000, stably circulated in the bloodstream with a half-life of ~1 day in a mouse, while an

6

unconjugated aptamer control showed a half-life of 5‒10 min.71 Note that simple conjugation

7

with PEG (or PEGylation) does not sufficiently protect siRNA from nuclease attack, and thus

8

chemically

9

phosphorothioate linkages, have been widely applied for conjugate preparation.

modified

(thus

stabilized)

siRNAs,

employing

2′-O-methylation

and

10

As an alternative to PEG, functional polymers comprising endosome-disrupting moieties

11

or targeting ligands have been developed. Amphipathic poly(vinyl ether) derivatives,72

12

poly(amido amine)s,73,74 and melittin-like polypeptides75 were reported as endosome-disrupting

13

backbone polymers. These polymers originally have positively charged groups, which

14

electrostatically interact with negatively charged endosomal membranes, eventually disrupting

15

them. However, these positively charged groups also disrupt cytoplasmic membranes and induce

16

cytotoxicity. Additionally, positively charged polymers are electrostatically associated with

17

siRNA and thus prohibit efficient covalent conjugation between them. To avoid these unwanted

18

behaviors, the positively charged groups, i.e., primary amines, can be masked with acidic pH- or

19

redox potential-responsive cleavable moieties, such as maleic acid amide and disulfide bonds,

20

respectively. Targeting ligands, PEG, and siRNA are conjugated to the backbone polymers via

21

such responsive moieties. In this way, acidic pH-responsive polymers can expose their positively

22

charged groups preferably in late endosomes/lysosomes, while simultaneously releasing other

23

components (e.g., targeting ligands and PEG).72 Similarly, redox potential-responsive polymers

24

can release siRNA in glutathione-rich cytoplasm. In the conjugate design, chemical bonds

25

between the polymer and siRNA need to be carefully selected to tolerate circulation in the

26

bloodstream. For instance, thiosuccinimide bonds, which are formed by the reaction of thiol and

27

alkyl maleimide, have been widely used for developing antibody-drug conjugates (ADCs). This

28

chemistry proceeds rapidly under physiological conditions and attains quantitative conjugation

29

without a large excess of reactant. However, thiosuccinimide bonds are reported to reversibly

30

cleave to form thiol and maleimide groups in plasma, which results in loss of drug from the ADC 17 ACS Paragon Plus Environment


1

during circulation,76 presumably diminishing the therapeutic efficacy. Shape (or topology) of

2

polymers (e.g., linear or cyclic polymers) influences their blood circulation property. In a

3

previous study, a cyclic polymer with MW of 115 kDa exhibited a blood half-life of 43.9 ± 2.2 h

4

in the beta phase while a linear polymer with MW of 114 kDa exhibited a blood half-life of 38.9

5

± 1.8 h in the beta phase.77,78 The shorter blood circulation property of the linear polymer might

6

be due to faster filtration from the glomerular basement membrane, compared with the cyclic

7

polymer. On the other hand, accumulation profiles of these two polymers in subcutaneous colon

8

tumors were not significantly different presumably because the difference in blood circulation

9

properties was marginal for passive tumor targeting.

10

One of the most well-known polymer-siRNA conjugates is Dynamic PolyConjugate, in

11

which PEGs, hepatocyte-targeting ligands, and siRNAs were conjugated to a poly(vinyl ether)

12

derivative backbone.72 Notably, siRNA was conjugated via a disulfide bond, which can be

13

cleaved in the reductive cytoplasm. PEGs and targeting ligands were attached via an acidic pH-

14

cleavable linkage, to be released in late endosomes/lysosomes. Dynamic PolyConjugate showed

15

a hydrodynamic diameter of 10 ± 2 nm with a negative ζ-potential, while the exact conjugation

16

rates of each moiety were not reported. Dynamic PolyConjugate with apolipoprotein B (apoB)-

17

targeted siRNA exhibited nearly 80% gene silencing for apoB mRNA at an siRNA concentration

18

of 50 nM in cultured primary hepatocytes. A single injection of Dynamic PolyConjugate (50 µg

19

of siRNA) into mice obtained ~90% gene silencing for apoB mRNA in the liver, and the RNAi

20

effects continued for 15 days. In a different study, brush type copolymers were synthesized from

21

5 and 10 kDa PEG with total MWs of 134 and 204 kDa, respectively. These brush type

22

copolymers were then conjugated to an internal position of a nucleic acid strand via click

23

conjugation (or strain-promoted azide-alkyne cycloaddition reaction) to reduce nuclease

24

accessibility.79 The high MW of the brush type PEG enabled tumor accumulation of the

25

conjugates through the EPR effect and protection of nucleic acid strands. This research used

26

short double-stranded DNA, instead of siRNA, as model sequences. The polymer-assisted

27

compaction of DNAs (pacDNAs) had hydrodynamic diameters of 17‒32 nm (depending on the

28

MW of the PEG chains) with slightly negative ζ-potentials close to neutral (−12 to −0.6 mV).

29

Although the blood circulation properties were not reported, the pacDNAs induced enhanced

30

accumulation in a mouse orthotopic breast tumor model.80 The large pacDNA with a diameter of 18 ACS Paragon Plus Environment

Page 18 of 41

Page 19 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

32 ± 8 nm more efficiently accumulated in the tumor, compared with the naked DNA control.

2

Interestingly, two different fluorophores were used to label brush PEG and DNA separately.

3

Fluorescence signals obtained from the two dyes were observed similarly in the tumor, indicating

4

that pacDNA was not degraded in the bloodstream and accumulated intact in the tumor. Whereas

5

the research did not investigate RNAi efficacy in the animal model, the pacDNA represented a

6

potential to formulate long-circulating PEG-siRNA conjugates (17‒32 nm in size) by using brush

7

PEGs with an extremely high MW. The effects of brush PEGs (or the shape of PEG) on cellular

8

internalization should be clarified in future studies to estimate the RNAi efficacy in this system.

9

The conjugation chemistry between the brush PEG and siRNA should also be optimized for PEG

10

detachment in the cytoplasm.

11 12

4.2. Electrostatically-associated siRNA delivery vehicles

13

Electrostatic associations between PEG-based block catiomers and siRNAs (or aniomers)

14

generate spontaneous assemblies, i.e., polyion complexes (PICs), through simple mixing of their

15

aqueous solutions. Thus, this formulation can avoid tedious conjugation and purification steps

16

associated with polymer-siRNA conjugate systems. As illustrated in Fig. 7A, the PIC formation

17

is subjected to two assembling processes; one is the formation of primary assemblies, termed

18

unit PICs (uPICs), with the minimal association number for charge neutralization and the other is

19

the secondary associations of uPICs to form multimolecular assemblies, e.g., micelles and

20

vesicles, above a critical association concentration (CAC).81–83 This section highlights the uPIC

21

formulation rather than conventional micellar vehicles, because of its appreciably smaller size

22

for enhanced tumor penetrability.

23

24 19 ACS Paragon Plus Environment


1

2 3

Figure 7. (A) Electrostatic association processes between PEG-b-catiomers and aniomers.86 (B)

4

Construction of uPICs by stoichiometric charge neutralization between PEG-PLL and siRNA.

5

(C) Chemical structures of FolA-PEG24-K(Stp4-C)2. (D) Schematic illustration for construction

6

of single siRNA nanocapules. Reprinted with permission from [87] and [88], respectively.

7

Copyright 2012, American Chemical Society.

8 9

The construction of siRNA-loaded uPICs was firstly accomplished by charge-

10

stoichiometric association between siRNA and PEG-b-poly(L-lysine) (PEG-PLL) with a well-

11

defined degree of polymerization (DP). The primary amines in PLL have a pKa of 10.5 and are

12

99% protonated at pH 7.4. Thus, the number of positively charged groups coupling with siRNA

13

phosphates is readily calculated from the DPPLL. A single siRNA molecule has a charge of −40,

14

which can be neutralized by a single PEG-PLL with DPPLL = 40 (+40 charges) or three PEG-

15

PLLs with DPPLL = 14 (+14 charges) below CAC [Fig. 7B].84,85 Interestingly, excess PEG-PLL

16

molecules with DPPLL = 40 did not participate in the electrostatic association with siRNA and

17

remained as free polymer; when 10 times charge-equivalent PEG-PLLs with DPPLL = 40 were 20 ACS Paragon Plus Environment

Page 20 of 41

Page 21 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

mixed with siRNAs, only 10% PEG-PLLs were bound to siRNAs, leaving 90% unbound PEG-

2

PLLs.84 Accordingly, the structures (or size and association number) of siRNA-loaded uPICs can

3

be precisely controlled by the MWPEG and DPPLL in block catiomer, which is a strong advantage

4

for construction of small siRNA delivery vehicles. A uPIC prepared from PEG-PLL (MWPEG =

5

42k and DPPLL = ~40) and siRNA exhibited a DH of 12.7 ± 0.6 nm,83 which may avoid the rapid

6

renal filtration. Another important criterion is the stability under physiological conditions. In this

7

regard, the ion pairs between PLL segment and siRNA were tolerable in 150 mM NaCl and 10%

8

fetal bovine serum-containing media,84 allowing for stable uPIC formation in the physiological

9

milieu. Note that a relatively high CAC for secondary association is required for selective

10

preparation of uPICs at a wide range of concentrations. Otherwise, the associates form

11

multimolecular structures (micelles or vesicles) with several tens nanometer in size [Fig. 7A]. A

12

previous study revealed that the rigid cylindrical structure of siRNA significantly elevated the

13

CAC values (>10 mg/mL) for the multimolecular assembly, compared with flexible single-

14

stranded RNA (ssRNA) ( 8.5, was selected for ion-pairing with

14

siRNA.89 The cross-linker B was degradable at pH < 6, allowing the release of siRNA in the

15

acidic compartments. The monomer C was a hydrophilic component facilitating colloidal

16

dispersion of the nanocapsule assembly in aqueous media. The resulting assembly showed an

17

average diameter of 20 nm in the TEM image. Interestingly, a dark core with a diameter around

18

5 nm was clearly observed within each assembly, which was probably tungsten-stained siRNA.

19

The assemblies were efficiently internalized by cultured cancer cells, probably due to their

20

positively charged surface (~12 mV in ζ-potential). As a result, the assemblies induced

21

significant luciferase gene silencing in cultured cancer cells, compared with a scramble siRNA-

22

loaded control. However, this study did not investigate RNAi efficacy in animal models.

23 24

4.3. Inorganic nanoparticle-based siRNA delivery vehicle

25

Owing to the precise synthesis of inorganic nanoparticles with various sizes and shapes,

26

size-controllable delivery vehicles have been constructed from inorganic nanoparticles as

27

templates. Inorganic nanoparticles, including gold nanoparticles (AuNPs), quantum dots, iron

28

oxide nanoparticles, and silica nanoparticles, have been tailored for incorporating siRNA

29

molecules on their surface via covalent (or coordinate) bonding. AuNPs with gold-sulfur (Au-S) 22 ACS Paragon Plus Environment

Page 22 of 41

Page 23 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

bonds are one of the most widely used formulations for oligonucleotide delivery due to their low

2

toxicity, high traceability, and ease in functionalization under mild conditions. The Au-S bond is

3

probably cleaved in glutathione-rich cytoplasm (~10 mM concentration),90,91 allowing the

4

delivery vehicles to release their payload siRNA. Besides Au-S bonding, covalent bonding (e.g.,

5

ZnS-thiol) and metal-phosphate coordination (e.g., Zr-phosphate) are also used for conjugation

6

of oligonucleotides onto inorganic nanoparticles, such as quantum dots, iron oxide nanoparticles,

7

and metal-organic framework nanoparticles.92–95 Additional conjugation of functional

8

components, such as PEG and peptide ligand, can further functionalize siRNA-conjugated

9

inorganic nanoparticles for enhanced colloidal stability, endosome-escapability, and targetability.

10

These conjugation techniques are in sharp contrast with the nanoparticle constructions by

11

electrostatic interactions, e.g., layer-by-layer technique, which often induce secondary

12

aggregations of nanoparticles, losing the size integrity.96,97

13

Spherical nucleic acids (SNAs) are known as a representative AuNP functionalized with

14

DNA or RNA containing thiol moieties [Fig. 8A].98,99 In this formulation, thiol-functionalized

15

siRNAs were densely decorated on a 13 nm-sized AuNP via Au-S bonding, and then,

16

oligo(ethylene glycol) or short PEG molecules were backfilled on the AuNP surface to increase

17

colloidal stability in the physiological milieu.100–102 The SNAs contained 37.8 ± 5.3 antisense

18

strands of siRNA per particle and had a DH of 33.6 ± 0.2 nm. They efficiently entered cultured

19

cells, mediated by scavenger receptors and lipid rafts, eliciting the enhanced RNAi, compared

20

with naked siRNA.100,103 Several parameters, including the spacer moiety between RNA and the

21

AuNP surface, the density of capped RNA, and the backfill molecules, affected the RNAi

22

efficacy of the SNAs.104,105 A pharmacokinetic study showed that 90% of the SNAs were quickly

23

eliminated from the bloodstream in the first 5 min after intravenous injection. The blood

24

circulation profile analyzed by a two-compartment model exhibited half-lives of ~1 min in the α

25

phase and ~8.5 h in the β phase.101 Interestingly, the SNAs were reported to penetrate the blood-

26

brain barrier and the blood-brain tumor barrier [Fig. 8B] and successfully delivered siRNA to

27

reduce the expression of Bcl2L12 mRNA in a glioblastoma.101

28

The short half-life of SNAs in the α phase is disadvantageous for tumor accumulation, as

29

aforementioned. Our previous study developed a long-circulating siRNA-loaded AuNP by

30

utilizing the 1:1 uPICs between siRNA and a thiolated PEG-PLL (MWPEG = 2k and DPPLL = 23 ACS Paragon Plus Environment


1

~40).84 The uPIC-conjugated AuNP (uPIC-AuNP) was prepared by a two-step bottom-up

2

approach [Fig. 8C]. siRNA-loaded uPICs were first prepared by the stoichiometric charge

3

neutralization between siRNA and thiolated PEG-PLL. Then, the thiolated uPICs were

4

conjugated on a 20 nm-sized AuNP template via Au-S bonding. The obtained uPIC-AuNPs had a

5

hydrodynamic diameter of 40 nm with a narrow size distribution and exhibited a prolonged

6

blood circulation property with a half-life of 30 min, probably due to the enhanced stealthiness

7

derived from the PEG shell. The sizes of uPIC-AuNPs were precisely controlled from 30 nm to

8

50 nm with a narrow size distribution (polydispersity index (PDI) in dynamic light scattering
200 nucleotide-long noncoding RNAs, which are coded by the

22

genome but are mostly not translated into proteins.114 LncRNAs are associated with various

23

types of cancer, and alternation in lncRNA expression promotes the tumorigenesis and

24

metastasis. LncRNA transcripts are degradable by using lncRNA-specific siRNAs and antisense

25

oligonucleotides (ASOs). The RISC-bound siRNAs can target the complementary mRNA only in

26

the cytoplasm, but RNase H-inducing ASOs can degrade mRNA in both the cytoplasm and the

27

nucleus.115 The cRGD-conjugated micelle with ASOs targeted for taurine-upregulated gene 1

28

(Tug1) lncRNA dramatically reduced the Tug1 lncRNA amounts and exerted the strong

29

antitumor effect for a patient-derived orthotopic brain tumor model.116 This preliminary result

30

demonstrates that lncRNAs are also promising targets in oligonucleotide therapeutics. 27 ACS Paragon Plus Environment


1 2

Acknowledgements

3

This work was financially supported by Center of Innovation (COI) program from Japan

4

Science and Technology Agency (JST) and Grants-in-Aid for Scientific Research (KAKENHI

5

Grant Numbers 17H02098 for KM and 18K18378 for HJK) from Ministry of Education Culture,

6

Sports, Science and Technology (MEXT). This work was also partially supported by the Project

7

for Cancer Research and Therapeutics Evolution (P-CREATE) and Basic Science and Platform

8

Technology Program for Innovative Biological Medicine from Japan Agency for Medical

9

Research and Development (AMED).

10 11

References

12

[1]

13

of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001,

14

411, 494.

15

[2]

16

interference. Nature 2009, 457, 405.

17

[3]

18

2014, 16, 10.

19

[4]

20

Current status and future directions. Cancer Treat. Rev. 2016, 50, 35.

21

[5]

22

allosterically control GTP affinity and effector interactions. Nature 2013, 503, 548.

23

[6]

24

insights to mechanism-based design. Nat. Rev. Drug Discov. 2016, 15, 771.

25

[7]

McCormick, F. KRAS as a therapeutic target. Clin. Cancer Res. 2015, 21, 1797.

26

[8]

Zorde Khvalevsky, E.; Gabai, R.; Rachmut, I. H.; Horwitz, E.; Brunschwig, Z.; Orbach,

27

A.; Shemi, A.; Golan, T.; Domb, A. J.; Yavin, E.; Giladi, H.; Rivkin, L.; Simerzin, A.; Eliakim,

28

R.; Khalaileh, A.; Hubert, A.; Lahav, M.; Kopelman, Y.; Goldin, E.; Dancour, A.; Hants, Y.;

Elbashir, S. M.; Harborth, J.; Lendeckel, W.; Yalcin, A.; Weber, K.; Tuschl, T. Duplexes

Jinek, M.; Doudna, J. A. A three-dimensional view of the molecular machinery of RNA

Fellmann, C.; Lowe, S. W. Stable RNA interference rules for silencing. Nat. Cell Biol.

Barata, P.; Sood, A. K.; Hong, D. S. RNA-targeted therapeutics in cancer clinical trials:

Ostrem, J. M.; Peters, U.; Sos, M. L.; Wells, J. A.; Shokat, K. M. K-Ras(G12C) inhibitors

Ostrem, J. M.; Shokat, K. M. Direct small-molecule inhibitors of KRAS: from structural


Page 28 of 41

Page 29 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

Arbel-Alon, S.; Abramovitch, R.; Shemi, A.; Galun, E. Mutant KRAS is a druggable target for

2

pancreatic cancer. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 20723.

3

[9]

4

J.; Kalluri, R. Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer.

5

Nature 2017, 546, 498.

6

[10]

7

Riggi, N.; Chi, A. S.; Cahill, D. P.; Nahed, B. V.; Curry, W. T.; Martuza, R. L.; Rivera, M. N.;

8

Rossetti, N.; Kasif, S.; Beik, S.; Kadri, S.; Tirosh, I.; Wortman, I.; Shalek, A. K.; Rozenblatt-

9

Rosen, O.; Regev, A.; Louis, D. N.; Bernstein, B. E. Reconstructing and reprogramming the

Kamerkar, S.; LeBleu, V. S.; Sugimoto, H.; Yang, S.; Ruivo, C. F.; Melo, S. A.; Lee, J.

Suvà, M. L.; Rheinbay, E.; Gillespie, S. M.; Patel, A. P.; Wakimoto, H.; Rabkin, S. D.;

10

tumor-propagating potential of glioblastoma stem-like cells. Cell, 2014, 157, 580.

11

[11]

12

Ahmed, A. U.; Balyasnikova, I. V.; Zhang, H. F.; Sun, C.; Langer, R.; Anderson, D. G.; Lesniak,

13

M. S. Multiplexed RNAi therapy against brain tumor-initiating cells via lipopolymeric

14

nanoparticle infusion delays glioblastoma progression. Proc. Natl. Acad. Sci. U. S. A. 2017, 114,

15

E6147.

16

[12]

17

targeted gene silencing. Chem. Biol. 2012, 19, 937.

18

[13]

19

delivery vehicles for cancer therapy. Adv. Drug Deliv. Rev. 2016, 104, 61.

20

[14]

21

impact of passive and active targeting in the era of modern cancer biology. Adv. Drug Deliv. Rev.

22

2014, 66, 2.

23

[15]

24

(and beyond). Chem. Rev. 2015, 115, 11043.

25

[16]

26

targets in cancer. Curr. Opin. Genet. Dev. 2005, 15, 102.

27

[17]

28

chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent

29

smancs. Cancer Res. 1986, 46, 6387.

30

[18]

31

Mech. Dis. 2016, 11, 251.

Yu, D.; Khan, O. F.; Suvà, M. L.; Dong, B.; Panek, W. K.; Xiao, T.; Wu, M.; Han, Y.;

Deleavey, G. F.; Damha, M. J. Designing chemically modified oligonucleotides for

Kim, H. J.; Kim, A.; Miyata, K.; Kataoka, K. Recent progress in development of siRNA

Bertrand, N.; Wu, J.; Xu, X.; Kamaly, N.; Farokhzad, O. C. Cancer nanotechnology: the

Lächelt, U.; Wagner, E. Nucleic acid therapeutics using polyplexes: a journey of 50 years

Baluk, P.; Hashizume, P.; McDonald, D. M. Cellular abnormalities of blood vessels as

Matsumura, Y.; Maeda, H. A new concept for macromolecular therapeutics in cancer

Park-Windhol, C.; D’Amore, P. A. Disorders of vascular permeability. Annu. Rev. Pathol.



1

[19]

Claesson-Welsh, L. Vascular permeability-the essentials. Ups. J. Med. Sci. 2015, 120,

2

135.

3

[20]

4

analysis of vascular permeability in mice using two-photon microscopy. Sci. Rep. 2013, 3, 1932.

5

[21]

6

transcellular transport pathways. Annu. Rev. Physiol. 2010, 72, 463.

7

[22]

8

Oncol. 2010, 7, 653.

9

[23]

Egawa, G.; Nakamizo, S.; Natsuaki, Y.; Doi, H.; Miyachi, Y.; Kabashima, K. Intravital

Komarova, Y.; Malik, A. B. Regulation of endothelial permeability via paracellular and

Jain, R. K.; Stylianopoulos, T. Delivering nanomedicine to solid tumors. Nat. Rev. Clin.

Dreher, M. R.; Liu, W.; Michelich, C. R.; Dewhirst, M. W.; Yuan, F.; Chilkoti, A. Tumor

10

vascular permeability, accumulation, and penetration of macromolecular drug carriers. J. Natl.

11

Cancer Inst. 2006, 98, 335.

12

[24]

13

Nat. Med. 2003, 9, 713.

14

[25]

15

Jain, R. K. Regulation of transport pathways in tumor vessels: role of tumor type and

16

microenvironment. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 4607.

17

[26]

18

Munster, P. N.; Campbell, K.; Gaddy, D. F.; Leonard, S. C.; Geretti, E.; Blocker, S. J.; Kirpotin,

19

D. B.; Moyo, V.; Wickham, T. J.; Hendriks, B. S. 64Cu-MM-302 positron emission tomography

20

quantifies variability of enhanced permeability and retention of nanoparticles in relation to

21

treatment response in patients with metastatic breast cancer. Clin. Cancer Res. 2017, 23, 4190.

22

[27]

23

R.; af Rosenschöld, P. M.; Kristensen, A. T.; Kjær, A.; Andresen, T. L. Positron emission

24

tomography based elucidation of the enhanced permeability and retention effect in dogs with

25

cancer using copper-63 liposomes. ACS Nano, 2015, 9, 6985.

26

[28]

27

N.; Higuchi, Y.; Konishi, S.; Kano, M. R.; Nishihara, H.; Miura, Y.; Nishiyama, N.; Kataoka, K.

28

Systemic targeting of lymph node metastasis through the blood vascular system by using size-

29

controlled nanocarriers. ACS Nano, 2015, 9, 4957.

30

[29]

31

K.; Milowsky, M. I.; Wobker, S. E.; Huang, L. The binding site barrier elicited by tumor-

McDonald, D. M.; Choyke, P. L. Imaging of angiogenesis: from macroscope to clinic.

Hobbs, S. K.; Monsky, W. L.; Yuan, F.; Roberts, W. G.; Griffith, L.; Torchilin, V. P.;

Lee, H.; Shields, A. F.; Siegel, B. A.; Miller, K. D.; Krop, I.; Ma, C. X.; LoRusso, P. M.;

Hansen, A. E.; Petersen, A. L.; Henriksen, J. R.; Boerresen, B.; Rasmussen, P.; Elema, D.

Cabral, H.; Makino, J.; Matsumoto, Y.; Mi, P.; Wu, H.; Nomoto, T.; Toh, K.; Yamada,

Miao, L.; Newby, J. M.; Lin, C. M.; Zhang, L.; Xu, F.; Kim, W. Y.; Forest, M. G.; Lai, S.


Page 30 of 41

Page 31 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

associated fibroblasts interferes disposition of nanoparticles in stroma-vessel type tumors. ACS

2

Nano, 2016, 10, 9243.

3

[30]

4

Drug Deliv. Rev. 2016, 99, 148.

5

[31]

6

molecular targeting on the intratumoral and subcellular distribution of polymeric nanoparticles.

7

Mol. Pharm. 2010, 7, 1195.

8

[32]

9

D. G.; Fukumura, D.; Jain, R. K.; Bawendi, M. G. A nanoparticle size series for in vivo

Dieterich, L. C.; Detmar, M. Tumor lymphangiogenesis and new drug development. Adv.

Lee, H.; Fonge, H.; Hoang, B.; Reilly, R. M.; Allen, C. The effects of particle size and

Popović, Z.; Liu, W.; Chauhan V. P.; Lee, J.; Wong, C.; Greytak, A. B.; Insin, N.; Nocera,

10

fluorescence imaging. Angew. Chem. Int. Ed. 2010, 49, 8649.

11

[33]

12

targeting efficiency of nanoparticles through design. Nano Lett. 2009, 9, 1909.

13

[34]

14

The role of micelle size in tumor accumulation, penetration, and treatment. ACS Nano, 2015, 9,

15

7195.

16

[35]

17

McDaid, K.; Kendrew, J.; Womack, C.; Wedge, S. R.; Barry, S. T. Tumor stromal architecture

18

can define the intrinsic tumor response to VEGF-targeted therapy. Clin. Cancer Res. 2013, 19,

19

6943.

20

[36]

21

Firpo, M. A.; Mulvihill, S. J. Phenotype and genotype of pancreatic cancer cell lines. Pancreas

22

2010, 39, 425.

23

[37]

24

A.; Kiyono, K.; Kaminishi, M.; Hirakawa, K.; Ouchi, Y.; Nishiyama, N.; Kataoka, K.; Miyazono,

25

K. Improvement of cancer-targeting therapy, using nanocarriers for intractable solid tumors by

26

inhibition of TGF-β signaling. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 3460.

27

[38]

28

Y.; Kano, M. R.; Miyazono, K.; Uesaka, M.; Nishiyama, N.; Kataoka, K. Accumulation of sub-

29

100 nm polymeric micelles in poorly permeable tumours depends on size. Nat. Nanotechnol.

30

2011, 6, 815.

Perrault, S. D.; Walkey, C.; Jennings, T.; Fischer, H. C.; Chan W. C. W. Mediating tumor

Wang, J.; Mao, W.; Lock, L. L.; Tang, J.; Sui, M.; Sun, W.; Cui, H.; Xu, D.; Shen, Y.

Smith, N. R.; Baker, D.; Farren, M.; Pommier, A.; Swann, R.; Wang, X.; Mistry, S.;

Deer, E. L.; González-Hernández, J.; Coursen, J. D.; Shea, J. E.; Ngatia, J.; Scaife, C. L.;

Kano, M. R.; Bae, Y.; Iwata, C.; Morishita, Y.; Yashiro, M.; Oka, M.; Fujii, T.; Komuro,

Cabral, H.; Matsumoto, Y.; Mizuno, K.; Chen, Q.; Murakami, M.; Kimura, M.; Terada,



1

[39]

McKee, T. D.; Grandi, P.; Mok, W.; Alexandrakis, G.; Insin, N.; Zimmer, J. P.; Bawendi,

2

M. G.; Boucher, Y.; Breakefield, X. O.; Jain, R. K. Degradation of fibrillar collagen in a human

3

melanoma xenograft improves the efficacy of an oncolytic herpes simplex virus vector. Cancer

4

Res. 2006, 66, 2509.

5

[40]

6

microenvironment to improve the permeation, retention, and antitumor effects of nanoparticles.

7

ACS Nano, 2017, 11, 4916.

8

[41]

9

Nat. Rev. Cancer, 2017, 17, 738.

Hu, K.; Miao, L.; Goodwin, T. J.; Li, J.; Liu, Q.; Huang, L. Quercetin remodels the tumor

Dewhirst, M. W.; Secomb, T. W. Transport of drugs from blood vessels to tumour tissue.

10

[42]

Dhillon, S.; Gill, K. In Clinical Pharmacokinetics, 1st ed.; Dhillon, S.; Kostrzewski, A.,

11

Eds.; Royal Pharmaceutical Society Publishing: Grayslake, IL, 2006; p1.

12

[43]

13

Miyata, K.; Oba, M.; Ogura, T.; Yamasaki, Y.; Nishiyama, N.; Yamasoba, T.; Kataoka, K.

14

Direct and instantaneous observation of intravenously injected substances using intravital

15

confocal micro-videography. Biomed. Opt. Express. 2010, 1, 1209.

16

[44]

17

Osada, A.; Takahashi, K.; Balkissoon, J.; Camp, A.; Masada, A.; Reitsma, D. J.; Bazhenova, L.

18

A. Phase Ib/II trial of NC-6004 (nanoparticle cisplatin) plus gemcitabine in patients with

19

advanced solid tumors. Clin. Cancer Res. 2018, 24, 43.

20

[45]

21

J.; Eatock, M.; Pearson, D. G.; Ottley, C. J.; Matsumura, Y.; Kataoka, K.; Nishiya, T. A phase I

22

clinical study of cisplatin-incorporated polymeric micelles (NC-6004) in patients with solid

23

tumours. Br. J. Cancer, 2011, 104, 593.

24

[46]

25

K.; Matsumura, Y.; Kataoka, K. Novel cisplatin-incorporated polymeric micelles can eradicate

26

solid tumors in mice. Cancer Res. 2003, 63, 8977.

27

[47]

28

N.; Kataoka, K. Bundled assembly of helical nanostructures in polymeric micelles loaded with

29

platinum drugs enhancing therapeutics efficiency against pancreatic tumor. ACS Nano, 2014, 8,

30

6724.

Matsumoto, Y.; Nomoto, T.; Cabral, H.; Matsumoto, Y.; Watanabe, S.; Christie, R. J.;

Subbiah, V.; Grilley-Olson, J. E.; Combest, A. J.; Sharma, N.; Tran, R. H.; Bobe, I.;

Plummer, R.; Wilson, R. H.; Calvert, H.; Boddy, A. V.; Griffin, M.; Sludden, J.; Tilby, M.

Nishiyama, N.; Okazaki, S.; Cabral, H.; Miyamoto, M.; Kato, Y.; Sugiyama, Y.; Nishio,

Mochida, Y.; Cabral, H.; Miura, Y.; Albertini, F.; Fukushima, S.; Osada, K.; Nishiyama,


Page 32 of 41

Page 33 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

[48]

Cabral, H.; Nishiyama, N.; Okazaki, S.; Koyama, H.; Kataoka K. Preparation and

2

biological

3

polymeric micelles. J. Control. Release, 2005, 101, 223.

4

[49]

5

J. Control. Release, 2014, 190, 465.

6

[50]

7

functional pathophysiology. Science, 2017, 357, eaal2379.

8

[51]

9

Deliv. Rev. 1998, 34, 93.

properties

of

dichloro(1,2-diaminocyclohexane)platinum(II)

(DACHPt)-loaded

Cabral, H.; Kataoka, K. Progress of drug-loaded polymeric micelles into clinical studies.

Augustin, H. G.; Koh, G. Y. Organotypic vasculature: from descriptive heterogeneity to

Takakura, Y.; Mahato, R. I.; Hashida, M. Extravasation of macromolecules. Adv. Drug

10

[52]

Nair, J. K.; Willoughby, J. L.; Chan, A.; Charisse, K.; Alam, M. R.; Wang, Q.; Hoekstra,

11

M.; Kandasamy, P.; Kel'in, A. V.; Milstein, S.; Taneja, N.; O'Shea, J.; Shaikh, S.; Zhang, L.; van

12

der Sluis, R. J.; Jung, M. E.; Akinc, A.; Hutabarat, R.; Kuchimanchi, S.; Fitzgerald, K.;

13

Zimmermann, T.; van Berkel, T. J.; Maier, M. A.; Rajeev, K. G.; Manoharan, M. Multivalent N-

14

acetylgalactosamine-conjugated siRNA localized in hepatocytes and elicits robust RNAi-

15

mediated gene silencing. J. Am. Chem. Soc. 2014, 136, 16958.

16

[53]

17

K.; Miyanishi, K.; Matsunaga, T.; Takayama, T.; Niitsu, Y. Resolution of liver cirrhosis using

18

vitamin A-coupled liposomes to deliver siRNA against a collagen-specific chaperone. Nat.

19

Biotechnol. 2008, 26, 431.

20

[54]

21

Jayaraman, M.; Rajeev, K. G.; Cantley, W. L.; Dorkin, J. R.; Butler, J. S.; Qin, L.; Racie, T.;

22

Spraque, A.; Fava, E.; Zeigerer, A.; Hope, M. J.; Zerial, M.; Sah, D. W.; Fitzgerald, K.; Tracy, M.

23

A.; Manoharan, M.; Koteliansky, V.; Fougerolles, A. d.; Maier, M. A. Targeted delivery of

24

RNAi therapeutics with endogenous and exogenous ligand-based mechanism. Mol. Ther. 2010,

25

18, 1357.

26

[55]

27

from live-cell and live-animal bioluminescent imaging. Nuclic Acids Res. 2006, 34, 322.

28

[56]

Schibler, U. Circadian rhythms. Liver regeneration clocks on. Science, 2003, 302, 234.

29

[57]

Jayaraman, M.; Ansell, S. M.; Mui, B. L.; Tam, Y. K.; Chen, J.; Du, X.; Butler, D.;

30

Eltepu, L.; Matsuda, S.; Narayanannair, J. K.; Rajeev, K. G.; Hafez, I. M.; Akinc, A.; Maier, M.

31

A.; Tracy, M. A.; Cullis, P. R.; Madden, T. D.; Manoharan, M.; Hope, M. J. Maximizing the

Sato, Y.; Murase, K.; Kato, J.; Kobune, M.; Sato, T.; Kawano, Y.; Takimoto, R.; Takada,

Akinc A.; Querbes, W.; De, S.; Qin, J.; Frank-Kamenetsky, M.; Jayaprakash, K. N.;

Bartlett, D. W.; Davis, M. E. Insights into the kinetics of siRNA-mediated gene silencing



1

potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angew. Chem. Int. Ed.

2

Engl. 2012, 51, 8529.

3

[58]

4

Stebbing, D.; Crosley, E. J.; Yaworski, E.; Hafez, I. M.; Dorkin, J. R.; Qin, J.; Lam, K.; Rajeev,

5

K. G.; Wong, K. F.; Jeffs, L. B.; Nechev, L.; Eisenhardt, M. L.; Jayaraman, M.; Kazem, M.;

6

Maier, M. A.; Srinivasulu, M.; Weinstein, M. J.; Chen, Q.; Alvarez, R.; Barros, S. A.; De, S.;

7

Klimuk, S. K.; Borland, T.; Kosovrasti, V.; Cantley, W. L.; Tam, Y. K.; Manoharan, M.;

8

Ciufolini, M. A.; Tracy, M. A.; de Fougerolles, A.; MacLachlan, I.; Cullis, P. R.; Madden, T. D.;

9

Hope, M. J. Rational design of cationic lipids for siRNA delivery. Nat. Biotechnol. 2010, 28, 172.

Semple, S. C.; Akinc, A.; Chen, J.; Sandhu, A. P.; Mui, B. L.; Cho, C. K.; Sah, D. W.;

10

[59]

Suhr, O. B.; Coelho, T.; Buades, J.; Pouget, J.; Conceicao, I.; Berk, J.; Schmidt, H.;

11

Waddington-Cruz, M.; Campistol, J. M.; Bettencourt, B. R.; Vaishnaw, A.; Gollob, J.; Adams, D.

12

Efficacy and safety of patisiran for familial amyloidotic polyneuropathy: a phase II multi-dose

13

study. Orphanet. J. Rare Dis. 2015, 10, 109.

14

[60]

15

Giese, K.; Kaufmann, J.; Khan, M.; Drevs, J. First-in-human phase I study of the liposomal RNA

16

interference therapeutic Atu027 in patients with advanced solid tumors. J. Clin. Oncol. 2014, 32,

17

4141.

18

[61]

19

Kaufmann, J.; Drevs, J. Phase I clinical development of Atu027, a siRNA formulation targeting

20

PKN3 in patients with advanced solid tumours. Int. J. Clin. Pharmacol. Ther. 2012, 50, 76.

21

[62]

22

K.; Fechtner, M.; Arnold, W.; Giese, K.; Klippel, A.; Kaufmann, J. A novel siRNA-lipoplex

23

technology for RNA interference in the mouse vascular endothelium. Gene Ther. 2006, 13, 1222.

24

[63]

25

J.; Paz-Ares, L.; Cho, D. C.; Infante, J. R.; Alsina, M.; Gounder, M. M.; Falzone, R.; Harrop, J.;

26

White, A. C.; Toudjarska, I.; Bumcrot, D.; Meyers, R. E.; Hinkle, G.; Svrzikapa, N.; Hutabarat,

27

R. M.; Clausen, V. A.; Cehelsky, J.; Nochur, S. V.; Gamba-Vitalo, C.; Vaishnaw, A. K.; Sah, D.

28

W.; Gollob, J. A.; Burris, H. A. 3rd. First-in-humans trial of an RNA interference therapeutic

29

targeting VEGF and KSP in cancer patients with liver involvement. Cancer Discov. 2013, 3, 406.

Schultheis, B.; Strumberg, D.; Santel, A.; Vank, C.; Gebhardt, F.; Keil, O.; Lange, C.;

Strumberg, D.; Schultheis, B.; Traugott, U.; Vank, C.; Santel, A.; Keil, O.; Giese, K.;

Santel, A.; Aleku, M.; Keil, O.; Endruschat, J.; Esche, V.; Fisch, G.; Dames, S.; Löffler,

Tabernero, J.; Shapiro, G. I.; LoRusso, P. M.; Cervantes, A.; Schwartz, G. K.; Weiss, G.


Page 34 of 41

Page 35 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

[64]

Landen, C. N. Jr; Chavez-Reyes, A.; Bucana, C.; Schmandt, R.; Deavers, M. T.; Lopez-

2

Berestein, G.; Sood, A. K. Therapeutic EphA2 gene targeting in vivo using neutral liposomal

3

small interfering RNA delivery. Cancer Res. 2005, 65, 6910.

4

[65]

5

Hutabarat, R.; Nochur, S.; Vaishnaw, A.; Gollob, J. Clinical proof of concept for a novel

6

hepatocyte-targeting GalNAc-siRNA conjugate. Mol. Ther. 2017, 25, 71.

7

[66]

8

nanoparticles. Int. J. Nanomedicine 2012, 7, 5577.

9

[67]

Zimmermann, T. S.; Karsten, V.; Chan, A.; Chiesa, J.; Boyce, M.; Bettencourt, B. R.;

Fröhlich, E. The role of surface charge in cellular uptake and cytotoxicity of medical

Williford, J. -M.; Santos, J. L.; Shyam, R.; Mao, H. -Q. Shape control in engineering of

10

polymeric nanoparticles for therapeutic delivery. Biomater. Sci. 2015, 3, 894.

11

[68]

12

Y. Y. The effects of polymeric nanostructure shape on drug delivery. Adv. Drug Deliv. Rev. 2011,

13

63, 1228.

14

[69]

15

Frangioni, J. V. Renal clearance of quantum dots. Nat. Biotechnol. 2007, 25, 1165.

16

[70]

17

M.; Hubbell, J. A.; Spencer, N. D. Poly(L-lysine)-g-poly(ethylene glycol) layers on metal oxide

18

surfaces: attachment mechanism and effects of polymer architecture on resistance to protein

19

adsorption. J. Phys. Chem. B. 2000, 104, 3298.

20

[71]

21

2010, 9, 537.

22

[72]

23

Bertin, S. L.; Reppen, T. W.; Chu, Q.; Blokhin, A. V.; Hagstrom, J. E.; Wolff, J. A. Dynamic

24

polyconjugates for targeted in vivo delivery of siRNA to hepatocytes. Proc. Natl. Acad. Sci. U. S.

25

A. 2007, 104, 12982.

26

[73]

27

Walsh, E.; Howell, B.; Sepp-Lorenzino, L.; Riley, S.; Patel, M.; Kemp, E.; Latham, A.; Leone,

28

A.; Soli, E.; Burke, R. S.; Carr, B.; Colletti, S. L.; Wang, W. Novel ensosomolytic poly(amido

29

amine) polymer conjugates for systemic delivery of siRNA to hepatocytes in rodents and

30

nonhuman primates. Bioconjug. Chem. 2014, 25, 896.

Venkataraman, S.; Hedrick, J. L.; Ong, Z. Y.; Yang, C.; Ee, P. L.; Hammond, P. T.; Yang,

Choi, H. S.; Liu, W.; Misra, P.; Tanaka, E.; Zimmer, J. P.; Itty Ipe, B.; Bawendi, M. G.;

Kenausis, G. L.; Vörös, J.; Elbert, D. L.; Huang, N.; Hofer, R.; Ruiz-Taylor, L.; Textor,

Keefe, A. D.; Pai, S.; Ellington A. Aptamers as therapeutics. Nat. Rev. Drug Discov.

Rozema, D. B.; Lewis, D. L.; Wakefield, D. H.; Wong, S. C.; Klein, J. J.; Roesch, P. L.;

Parmar, R. G.; Poslusney, M.; Busuek, M.; Williams, J. M.; Garbaccio, R.; Leander, K.;



1

[74]

Barrett, S. E.; Burke, R. S.; Abrams, M. T.; Bason, C.; Busuek, M.; Carlini, E.; Carr, B.

2

A.; Crocker, L. S.; Fan, H.; Garbaccio, R. M.; Guidry, E. N.; Heo, J. H.; Howell, B. J.; Kemp, E.

3

A.; Kowtoniuk, R. A.; Latham, A. H.; Leone, A. M.; Lyman, M.; Parmar, R. G.; Patel, M.;

4

Pechenov, S. Y.; Pei, T.; Pudvah, N. T.; Raab, C.; Riley, S.; Sepp-Lorenzino, L.; Smith, S.; Soli,

5

E. D.; Staskiewicz, S.; Stern, M.; Truong, Q.; Vavrek, M.; Waldman, J. H.; Walsh, E. S.;

6

Williams, J. M.; Young, S.; Colletti, S. L. Development of a liver-targeted siRNA delivery

7

platform with a broad therapeutic window utilizing biodegradable polypeptide-based polymer

8

conjugates. J. Control. Release, 2014, 183, 124.

9

[75]

Wooddell, C. I.; Rozema, D. B.; Hossbach, M.; John, M.; Hamilton, H. L.; Chu, Q.;

10

Hegge, J. O.; Klein, J. J.; Wakefield, D. H.; Oropeza, C. E.; Deckert, J.; Roehl, I.; Jahn-Hofmann,

11

K.; Hadwiger, P.; Vornlocher, H. P.; McLachlan, A.; Lewis, D. L. Hepatocyte-targeted RNAi

12

therapeutics for the treatment of chronic hepatitis B virus infection. Mol. Ther. 2013, 21, 973.

13

[76]

14

Balasubramanian, C. L.; Duniho, S. M.; Leiske, C. I.; Li, F.; Senter, P. D. Self-hydrolyzing

15

maleimides improve the stability and pharmacological properties of antibody-drug conjugates.

16

Nat. Biotechnol. 2014, 32, 1059.

17

[77]

18

Dependence of pharmacokinetics and biodistribution on polymer architecture: effect of cylic

19

versus linear polymers. J. Am. Chem. Soc. 2009, 131, 3842.

20

[78]

21

pharmacokinetics: differences between cyclic and linear PEGylated poly(acrylic acid) comb

22

polymers. J. Control. Release 2009, 140, 203.

23

[79]

24

nuclease stability and the gene silencing efficacy of brush-architectured poly(ethylene glycol)-

25

DNA conjugates. J. Am. Chem. Soc. 2017, 139, 10605.

26

[80]

27

Providing oligonucleotides with steric selectivity by brush-polymer-assisted compaction. J. Am.

28

Chem. Soc. 2015, 137, 12466.

29

[81]

30

from oppositely charged block copolymers. Science, 1999, 283, 65.

Lyon, R. P.; Setter, J. R.; Bovee, T. D.; Doronina, S. O.; Hunter, J. H.; Anderson, M. E.;

Nasongkla, N.; Chen, B.; Macaraeg, N.; Fox, M. E.; Fréchet, J. M. J.; Szoka, F. C.

Chen, B.; Jerger, K.; Fréchet, J. M. J.; Szoka, F. C. The influence of polymer topology on

Jia, F.; Lu, X.; Wang, D.; Cao, X.; Tan, X.; Lu, H.; Zhang, K. Depth-profiling the

Lu, X.; Tran, T. H.; Jia, F.; Tan, X.; Davis, S.; Krishnan, S.; Amiji, M. M.; Zhang, K.

Harada, A.; Kataoka, K. Chain length recognition: core-shell supramolecular assembly


Page 36 of 41

Page 37 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

[82]

Anraku, Y.; Kishimura, A.; Yamasaki, Y.; Kataoka K. Living unimodal growth of

2

polyion complex vesicles via two-dimensional supramolecular polymerization. J. Am. Chem. Soc.

3

2013, 135, 1423.

4

[83]

5

Nishiyama, N.; Miyata, K.; Kataoka, K. Influence of RNA strand rigidity on polyion complex

6

formation with block catiomers. Macromol. Rapid Commun. 2016, 37, 486.

7

[84]

8

Pittella, F.; Christie, R. J.; Toh, K.; Matsumoto, Y.; Nishiyama, N.; Miyata, K.; Kataoka, K.

9

Precise engineering of siRNA delivery vehicles to tumors using polyion complexes and gold

Hayashi, K.; Chaya, H.; Fukushima, S.; Watanabe, S.; Takemoto, H.; Osada, K.;

Kim, H. J.; Takemoto, H.; Yi, Y.; Zheng, M.; Maeda, Y.; Chaya, H.; Hayashi, K.; Mi, P.;

10

nanoparticles. ACS Nano, 2014, 8, 8979.

11

[85]

12

Monomolecular

13

Biomacromolecules, 2008, 9, 724.

14

[86]

15

design, characterization and biological significance. Adv. Drug Deliv. Rev. 2001, 47, 113.

16

[87]

17

Hadwiger, P.; Vornlocher, H. P.; Wagner, E. Nanosized multifunctional polyplexes for receptor-

18

mediated siRNA delivery. ACS Nano, 2012, 6, 5198.

19

[88]

20

enhanced RNAi delivery. J. Am. Chem. Soc. 2012, 134, 13542.

21

[89]

22

K. Enhanced stability and gene silencing ability of siRNA-loaded polyion complexes formulated

23

form polyaspartamide derivatives with a repetitive array of amino groups in the side chain.

24

Biomaterials, 2012, 33, 2770.

25

[90]

26

Acta. 2015, 1850, 1527.

27

[91]

28

Acta. 2012, 1823, 1767.

29

[92]

30

delivery and silencing in tumors. Nat. Med. 2007, 13, 372.

DeRouchey, J.; Schmidt, C.; Walker, G. F.; Koch, C.; Plank, C.; Wagner, E.; Rädler, J. O. assembly

of

siRNA

and

poly(ethylene

glycol)-peptide

copolymers.

Kataoka, K.; Harada, A.; Nagasaki, Y. Block copolymer micelles for drug delivery:

Dohmen, C.; Edinger, D.; Fröhlich, T.; Schreiner, L.; Lächelt, U.; Troiber, C.; Rädler, J.;

Yan, M.; Liang, M.; Wen, J.; Liu, Y.; Lu, Y.; Chen, I. S. Single siRNA nanocapsules for

Suma, T.; Miyata, K.; Ishii, T.; Uchida, S.; Uchida, H.; Itaka, K.; Nishiyama, N.; Kataoka

Hansen, J. M.; Harris, C. Glutathione during embryonic development. Biochim. Biophys.

Circu, M. L.; Aw, T. Y. Glutathione and modulation of cell apoptosis. Biochim Biophys.

Medarova, Z.; Pham, W.; Farrar, C.; Petkova, V.; Moore, A. In vivo imaging of siRNA



1

[93]

Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.; Sundar, V. C.; Mikulec, F.

2

V.; Bawendi, M. G. Self-assembly of CdSe-ZnS quantum dot bioconjugates using an engineered

3

recombinant protein. J. Am. Chem. Soc. 2000, 122, 12142.

4

[94]

5

C. A. General and direct method for preparing oligonucleotides-functionalized matal-organic

6

framework nanoparticles. J. Am. Chem. Soc. 2017, 139, 9827.

7

[95]

8

metal organic framework (MOF) nanoparticle conjugates. J. Am. Chem. Soc. 2014, 136, 7261.

9

[96]

Wang, S.; McGuirk, C. M.; Ross, M. B.; Wang, S.; Chen, P.; Xing, H.; Liu, Y.; Mirkin,

Morris, W.; Briley, W. E.; Auyeung, E.; Cabezas, M. D.; Mirkin C. A. Nucleic acid-

Lee, M. Y.; Park, S. J.; Park, K.; Kim, K. S.; Lee, H.; Hahn, S. K. Target-specific gene

10

silencing of layer-by-layer assembled gold-cysteamine/siRNA/PEI/HA nanocomplex. ACS Nano,

11

2011, 5, 6138.

12

[97]

13

siRNA-coated gold nanoparticles. Small, 2011, 7, 364.

14

[98]

15

A. Oligonucleotide-modified gold nanoparticles for intracellular gene regulation. Science, 2006,

16

312, 1027.

17

[99]

18

134, 1376.

19

[100] Giljohann D. A.; Seferos, D. S.; Prigodich, A. E.; Patel, P. C.; Mirkin, C. A. Gene

20

regulation with polyvalent siRNA-nanoparticle conjugates. J. Am. Chem. Soc. 2009, 131, 2072.

21

[101] Jensen, S. A.; Day, E. S.; Ko, C. H.; Hurley, L. A.; Luciano, J. P.; Kouri, F. M.; Merkel,

22

T. J.; Luthi, A. J.; Patel, P. C.; Cutler, J. I.; Daniel, W. L.; Scott, A. W.; Rotz, M. W.; Meade, T.

23

J.; Giljohann, D. A.; Mirkin, C. A.; Stegh, A. H. Spherical nucleic acid nanoparticle conjugates

24

as an RNAi-based therapy for glioblastoma. Sci. Transl. Med. 2013, 5, 209ra152.

25

[102] Chinen, A. B.; Ferrer, J. R.; Merkel, T. K.; Mirkin, C. A. Relationships between

26

poly(ethylene glycol) modifications on RNA-spherical nucleic acid conjugates and cellular

27

uptake and circulation time. Bioconjugate Chem. 2016, 27, 2715.

28

[103] Patel, P. C.; Giljohann, D. A.; Daniel, W. L.; Zheng, D.; Prigodich, A. E.; Mirkin, C. A.

29

Scavenger receptors mediate cellular uptake of polyvalent oligonucleotide-functionalized gold

30

nanoparticles. Bioconjug. Chem. 2010, 21, 2250.

Lee, S. K.; Han, M. S.; Asokan, S.; Tung, C. H. Effective gene silencing by multilayered

Rosi, N. L.; Giljohann, D. A.; Thaxton, C. S.; Lytton-Jean, A. K.; Han, M. S.; Mirkin, C.

Cutler, J. I.; Auyeung, E.; Mirkin, C. A. Spherical nucleic acids. J. Am. Chem. Soc. 2012,


Page 38 of 41

Page 39 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

[104] Barnaby, S. N.; Perelman, G. A.; Kohlstedt, K. L.; Chinen, A. B.; Schatz, G. C.; Mirkin,

2

C. A. Design considerations for RNA spherical nucleic acids (SNAs). Bioconjug. Chem. 2016,

3

27, 2124.

4

[105] Chinen, A. B.; Guan, C. M.; Ko, C.H.; Mirkin, C. A. The impact of protein corona

5

formation on the macropharge cellular uptake and biodistribution of spherical nucleic acids.

6

Small, 2017, 13, 1603847.

7

[106] Yi, Y.; Kim, H. J.; Mi, P.; Zheng, M.; Takemoto, H.; Toh, K.; Kim, B. S.; Hayashi, K.;

8

Naito, M.; Matsumoto, Y.; Miyata, K.; Kataoka, K. Targeted systemic delivery of siRNA to

9

cervical cancer model using cyclic RGD-installed unimer polyion complex-assembled gold

10

nanoparticles. J. Control. Release, 2016, 244, 247.

11

[107] Liu, G.; Choi, K. Y.; Bhirde, A.; Swiercaewska, M.; Yin, J.; Lee, S. W.; Park, J. H.;

12

Hong, J. I.; Xie, J.; Niu, G.; Kiesewetter, D. O.; Lee, S.; Chen, X. Sticky nanoparticles: a

13

platform for siRNA delivery by a bis(zinc(II)dipicolylamine)-functionalized, self-assembled

14

nanoconjugate. Angew. Chem. Int. Ed. 2012, 51, 445.

15

[108] Choi, D. W.; Koh, J. Y. Zinc and brain injury. Annu. Rev. Neurosci. 1998, 21, 347.

16

[109] Phillips, E.; Penate-Medina, O.; Zanzonico, P. B.; Carvajal, R. D.; Mohan, P.; Ye, Y.;

17

Humm, J.; Gönen, M.; Kalaigian, H.; Schöder, H.; Strauss, H. W.; Larson, S. M.; Wiesner, U.;

18

Bradbury, M. S. Clinical translation of an ultrasmall inorganic optical-PET imaging nanoparticle

19

probe. Sci. Transl. Med. 2014, 6, 260ra149.

20

[110] Ranneh, A. H.; Takemoto, H.; Sakuma, S.; Awaad, A.; Nomoto, T.; Mochida, Y.; Matsui,

21

M.; Tomoda, K.; Naito, M.; Nishiyama, N. An ethylenediamine-based switch to render the

22

polyzwitterion cationic at tumorous pH for effective tumor accumulation of coated nanometerials.

23

Angew. Chem. Int. Ed. 2018, doi: 10.1002/anie.201801641.

24

[111] Li, J.; Yu, X.; Wang, Y.; Yuan, Y.; Xiao, H.; Cheng, D.; Shuai, X. A reduction and pH

25

dual-sensitive polymeric vector for long-circulating and tumor-targeted siRNA delivery. Adv.

26

Mater. 2014, 26, 8217.

27

[112] Tangsangasaksri, M.; Takemoto, H.; Naito, M.; Maeda Y.; Sueyoshi, D.; Kim, H. J.;

28

Miura, Y.; Ahn, J.; Azuma, R.; Nishiyama, N.; Miyata, K.; Kataoka, K. siRNA-loaded polyion

29

complex micelle decorated with charge-conversional polymer tuned to undergo stepwise

30

response to intra-tumoral and intra-endosomal pHs for exerting enhanced RNAi efficacy.

31

Biomacromolecules 2016, 17, 246. 39 ACS Paragon Plus Environment


1

[113] Miyata, K.; Nishiyama, N.; Kataoka, K. Rational design of smart supramolecular

2

assemblies for gene delivery: chemical challenges in the creation of artificial viruses. Chem. Soc.

3

Rev. 2012, 41, 2562.

4

[114] Bhan, A.; Soleimani, M.; Mandal, S. S. Long noncoding RNA and cancer: a new

5

paradigm. Cancer Res. 2017, 77, 3965.

6

[115] Liang, X. H.; Sun, H.; Nichols, J. G.; Crooke, S. T. RNase H1-dependent antisense

7

oligonucleotides are robustly active in directing RNA cleavage in both the cytoplasm and the

8

nucleus. Mol. Ther. 2017, 25, 2075.

9

[116] Katsushima, K.; Natsume, A.; Ohka, F.; Shinjo, K.; Hatanaka, A.; Ichimura, N.; Sato, S.;

10

Takahashi, S.; Kimura, H.; Totoki, Y.; Shibata, T.; Naito, M.; Kim, H. J.; Miyata, K.; Kataoka,

11

K.; Kondo, Y. Targeting the Notch-regulated non-coding RNA Tug1 for glioma treatment. Nat.

12

Commun. 2016, 7, 13616.

13


Page 40 of 41

Page 41 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Biomacromolecules

Table of Contents Graphic

2


Small Delivery Vehicles of siRNA for Enhanced Cancer Targeting

Recommend Documents