Nanotoxicity: An Obstacle to siRNA-Based Nanomedicine

Table 1. Clinical trials of locally delivered siRNA therapeutics.
siRNA	Carrier	Administration route	Year started	Phase	Disease	Notes	ClinicalTrials.gov identifier
Bevasiranib (Cand5)	NC	Intravitreal	2004	I	AMD	First RNAi clinical trial	NCT00722384
			2005	II	AMD		NCT00259753
			2006	II	DME		NCT00306904
			2007	III	AMD	Terminated due to lack of efficacy	NCT00499590
			2009	III	AMD	Withdrawn	NCT00557791
Sirna-027 (AGN211745)	NC	Intravitreal	2004	I	AMD		NCT00363714
			2007	II		Terminated due to lack of efficacy	NCT00395057
PF-04523655 (REDD14NP, RTP801I-14)	NC	Intravitreal	2007	I	AMD		NCT00725686
			2008	II	DME	Terminated due to lack of efficacy	NCT00701181
			2009	II	AMD		NCT00713518
			2012^†	II	DME		NCT01445899
QPI-1002 (I5NP)	NC	iv. injection	2007	I	Acute kidney injury post cardiac surgery		NCT00554359
			2008	I		Terminated due to lack of participants	NCT00683553
			2008^†	I/II	Delayed graft function post kidney transplant		NCT00802347
QPI-1007	NC	Intravitreal	2010	I	Optic nerve atrophy, NAION		NCT01064505
ALN-RSV	NC	Intranasal	2007	II	RSV infection		NCT00496821
		Nebulization	2008	II	RSV infection in lung transplant patients		NCT00658086
			2010	II			NCT01065935
TD101	NC	Local injection	2008	I	Pachyonychia congenita	1 patient	NCT00716014
SYL040012	NC	Ophthalmic drop	2009	I	Ocular hypertension, glaucoma		NCT00990743
			2010	I/II			NCT01227291
			2012	II			NCT01739244
SYL1001	NC	Ophthalmic drop	2011	I	Ocular pain, dry eye syndrome		NCT01438281
			2012^†	I/II			NCT01776658
siG12D	LODER®	Implantation in tumor	2011^†	I	Pancreatic cancer		NCT01188785
			2012^†	II	Pancreatic cancer		NCT01676259

Details of each trial can be found on www.clinicaltrials.gov.
^†Ongoing trials.
AMD: Age-related macular degeneration; DME: Diabetic macular edema; iv.: Intravenous; LODER: Local Drug EluteR; NAION: Nonarteritic anterior ischemic optic neuropathy; NC: No carrier used; RSV: Respiratory syncytial virus.

Table 2. Clinical trials of systemically delivered siRNA therapies.
Drug name	siRNA	Carrier	Administration route	Year started	Phase	Disease	Notes	ClinicalTrials.gov identifier
CALAA-01	C05C	Targeted CDP^†	iv. infusion	2008^‡	I	Solid tumor	First siRNA trial utilizing nanocarrier	NCT00689065
Atu027	siRNA vs PKN3	Liposome^§	iv. infusion	2009	I	Solid tumor		NCT00938574
				2013^‡	I/II	Pancreatic cancer		NCT01808638
ALN-VSP	siRNA vs VEGF and KSP	LNP	iv. infusion	2009	I	Primary and secondary liver cancer		NCT00882180
				2010	I	Solid tumor	To assess long-term safety in patients who completed the previous study	NCT01158079
ALN-TTR	siRNA vs TTR	LNP	iv. infusion	2010	I	ATTR^#		NCT01148953
				2012	I			NCT01559077
				2012^‡	II			NCT01617967
ALN-PCS	siRNA vs PCSK9	LNP	iv. infusion	2011	I	Hypercholesterolemia		NCT01437059
TKM-ApoB (ApoB SNALP, PRO-040201)	siRNA vs ApoB	LNP	iv. infusion	2009	I	Hypercholesterolemia	Terminated due to immune stimulation potential	NCT00927459
TKM-PLK1 (PLK1 SNALP, TKM-080301)	siRNA vs PLK1	LNP	iv. infusion	2010	I	Solid tumor		NCT01262235
				2011	I	Primary and secondary liver cancer		NCT01437007
TKM-Ebola (Ebola SNALP, TKM-100201)	siRNA vs ZEBOV L polymerase, VP24 and VP35	LNP	iv. infusion	2012	I	Ebola virus infection		NCT01518881
ND-L02-s0201	siRNA vs HSP47	Vitamin A- coupled LNP	iv. injection	2013^‡	I	Fibrosis		NCT01858935
siRNA-EphA2-DOPC	siRNA vs EphA2	DOPC^¶	iv. infusion	2013^‡	I	Solid tumor		NCT01591356

Details of each trial can be found on www.clinicaltrials.gov.
^†Consists of cyclodextrin-containing polymer CAL101, stabilizing agent adamantine-PEG, and targeting agent adamantine-PEG-transferrin for transferrin receptor.
^‡Ongoing trials.
^§Consists of cationic lipid AtuFECT01, neutral helper lipid 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, and PEGylated lipid DSPE-methoxypolyethylene PEG.
^¶1,2-dioleoyl-sn-glycero-3-phosphatidylcholine neutral liposome.
^#Transthyretin-mediated amyloidosis. ATTR: Transthyretin-mediated amyloidosis; DOPC: 1,2-dioleoyl-sn-glycero-3-phosphocholine; iv.: Intravenous; LNP: Lipid nanoparticle; TTR: Transthyretin.

Table 3. Key factors that modify nanocarrier toxicity.
Factor	Example	Ref.
Shape	Long, needle-shaped carbon nanotubes are more cytotoxic than shorter ones	[97]
Size	Intermediate size (e.g., 8–37 nm) gold NPs caused higher in vivo toxicity in mice than very small (e.g., 3 nm) and large (e.g., 100 nm) ones	[89]
Surface charge	Neutral gold NPs caused cell death through necrosis, whereas charged nanoparticles induced apoptotic cell death	[25]
Surface hydrophobicity/hydrophilicity	Hydrophobic modifications of NPs caused a severe inflammatory response	[106]
Nanocarrier ingredients	Nanocarriers made of linear, low-molecular-weight PEIs caused less inflammatory responses than those made of branched, high-molecular-weight PEIs	[26]
Route of exposure	Direct systemic administration is associated with the highest risk of systemic toxicity due to accumulation of nanomaterials in the vital organs, such as the liver and kidneys. Other routes (e.g., topical, inhaled or oral) generally cause local toxicity at the sites of exposure, but can also lead to significant systemic toxicity if the nanomaterials are small enough (<100 nm) to penetrate the barrier structures (e.g., skin and GI tract)	[27]

NP: Nanoparticle; PEI: Polyethylenimine.

Table 4. Common types of toxicity mediated by siRNA nanocarriers.
Cause	Outcome
Significant direct damage to cell membrane integrity	Trigger acute cell death by necrosis and cell lysis within a short time (e.g., hours) after nanocarrier exposure, may initiate inflammatory responses to surrounding tissues
Significant direct damage to critical cellular components, for example, chromosomes and mitochondria	Trigger apoptotic cell death, typically a few days after nanocarrier exposure
Oxidative stress induced	Induce production of ROS. Moderate level of ROS causes DNA damages and lipid peroxidation; high level of ROS may trigger apoptosis. ROS generation is related to the nanomaterial, nanoparticle surface area and impurities in the nanodevices
Nanocarriers overloading the lysosomes, for example, owing to poor biodegradability or inhibitive effects on lysosomal enzymes	Leads to accumulation of visible autophagic vacuoles and autophagic dysfunction. Significant autophagy increases risk of apoptotic cell death
Altered gene expression (i.e., toxicogenomics)	From subtle disturbance of normal cell functions and phenotypes to measurable reduction in cell proliferation and even cell death
Genotoxicity	Certain nanocarriers (e.g., carbon nanotubes) can localize with the cell nucleus and induce DNA strand breakage
Immunogenic response stimulated	Reduce compatibility of the nanocarriers with the blood, mild-to-severe (anaphylaxis) hypersensitivity reactions, inflammatory responses (e.g., cytokine production), anaphylaxis, suppressed body defense to infection and cancer cell spreading, and myelosuppression (this effect might be exploited for therapy) Nanocarriers that are poorly designed and made of unfavorable materials may increase the risk of Toll-like receptor activation by siRNAs during the endosomal escape process, triggering cytokine production

ROS: Reactive oxygen species.

Table 5. Summary of strategies for reducing siRNA carrier nanotoxicity.
Strategy	Example	Ref.
Optimization of carrier materials
Use linear, low-molecular-weight polymer	Linear PEI induced less proinflammatory cytokines than the branched counterparts	[26]
Use polymer with higher biodegradability	siRNA nanocarriers made of biodegradable poly(amino esters) demonstrated significantly higher transfection efficiency and lower toxicity to lung cells when compared with 25-kDa PEI	[54]
Use well-established natural polymer	Human lung cancer cells transfected with a chitosan nanosystem demonstrated higher viability than when using a commercial lipid transfection agent	[61]
Use bioreducible polymer	Nanocarriers prepared by cross-linking low-molecular-weight linear poly(ethylenimine sulfide) quickly degraded in the reductive intracellular environment and produced degradants that were essentially nontoxic (cell survival 98.69%)	[104]
Engineer cationic lipids based on structure–toxicity analysis	Lipids containing biodegradable linker had lower inherent cytotoxicity	[79]
Optimization of physical properties
Screen for right size	Gold NPs with the smallest sizes (3 and 5 nm) and the largest sizes (50 and 100 nm) are nontoxic, but the intermediate sizes (8–37 nm) had lethal effects on mice	[89]
Masking the cationic charges	PAMAM dendrimer was engineered to keep the cationic charges inside the carrier instead of on the surface, resulting in significantly reduced dendrimer toxicity while improving the in vivo tumor-targeting properties when conjugated with LHRH	[90]
Increase surface hydrophilicity with PEGlyation	After pulmonary administration, it was shown that hydrophilic modifications of PEI nanocarriers with PEG grafting induced fewer proinflammatory effects without depleting macrophages and disrupting the epithelial/endothelial barrier in the lungs	[106]
Alternative delivery routes	Spherical nucleic acid nanoparticle conjugates applied topically did not cause clinical or histological signs of toxicity or trigger cytokine activation in mouse blood or tissue samples	[107]
Low-dose approach based on combinatorial synthesis and screening	Highly potent lipidoid-based siRNA nanoparticles allowed low-dose treatment that was well tolerated in mice	[108]
Hybrid nanocarrier made of two or more classes of materials	Solid lipids in lipid–polymer hybrid nanocarriers reduced the toxicity of PEI components in human breast epithelial cells as indicated by less membrane damage, improved mitochondrial function, reduced reactive oxidative species production and lower caspase-3 activity	[30]
Active targeting
EGFR targeting	Asymmetric liposome particles showed no cell toxicity	[114]
Folate receptor targeting	Folate-PEG-grafted siRNA nanocarriers demonstrated favorable cytotoxicity, transfection efficiency and cancer cell specificity compared with PEG-grafted nanocarriers	[115]
LHRH peptide-based targeting	NPs containing superparamagnetic iron oxide and poly(propyleneimine) dendrimer functionalized with LHRH peptide and PEG demonstrated enhanced serum stability, specific cellular uptake and low toxicity in A549 cells	[90]
Cell-penetrating peptide-based targeting	Chitosan-based siRNA nanocarriers functionalized with PEG and the cell-penetrating peptide TAT exhibited maximum cell transfection ability and very low cytotoxicity in Neuro2a neuroblastoma cells	[116]

NP: Nanoparticle; PAMAM: Polyamidoamine; PEI: Polyethylenimine.

Table 6. *In vitro* immunology assays used in the Nanotechnology Characterization Laboratory.
Test	Cells/materials	Goal
Blood contact properties
Analysis of hemolytic properties of NPs	Normal human whole blood	Colorimetric determination of hemoglobin released from red blood cells after NP exposure
Analysis of platelet aggregation	Sodium citrate human whole blood	Screening of potential anticoagulant or thrombogenic properties of NPs
Analysis of NP interaction with plasma proteins by 2D PAGE	Sodium citrate human whole blood	Evaluate protein adsorption on NPs that increases risk of opsonization by macrophages
Qualitative analysis of total complement activation by western blot	Sodium citrate human plasma	Qualitative determination of total complement activation by western blot analysis
Quantitative analysis of complement activation	Sodium citrate human plasma	Quantitative determination of complement activation by an enzyme immunoassay
Coagulation assay	Sodium citrate human whole blood	Assess the NP effects on plasma coagulation time
Cell-based assays
Mouse granulocyte macrophage colony-forming unit assay	Hematopoietic stem cells	Assess the effects of NPs related to myelosuppression
Leukocyte proliferation assay	Human lymphocytes	Measure NP effects on lymphocyte proliferation
Detection of nitric oxide production by RAW 264.7 macrophage cell line	RAW 264.7 murine macrophages	Measure effect of NPs on production of cytotoxic nitric oxide by macrophages
Measurement of NP effects on cytotoxic activity of NK cells	NK92 cells/hepatocarcinoma HepG2 cells	Study NP's effect on the cytotoxicity of NK cells
Analysis of NP effects on maturation of monocyte derived DCs	Human peripheral blood mononuclear cells	Measure NP effects on DC maturation in response to inflammatory stimuli, such as endotoxin
In vitro induction of leukocyte procoagulant activity by NPs	HL-60 cells	Assess the ability of NPs to induce leukocyte procoagulant activity
Oxidative stress
Hep G2 hepatocyte glutathione assay	Hep G2 cells	Analyze NP effects on reduced and oxidized glutathione
Hep G2 hepatocyte lipid peroxidation assay	Hep G2 cells	Analyze NP effects on formation of lipid peroxidation products such as malondialdehyde
Hepatocyte primary ROS assay	Primary hepatocytes	Measure NP effects ROS generation by fluorescence spectrophotometry
Cytotoxicity (necrosis and apoptosis)
Hep G2 hepatocarcinoma cytotoxicity assay	Hep G2 cells	Measure NP cytotoxic effects on liver cells by MTT and LDH release assay
Hep G2 hepatocarcinoma apoptosis assay (caspase 3 activation)	Hep G2 cells	Measure NP apoptotic effects (caspase 3 activation) on liver cells
Hep G2 hepatocarcinoma homogeneous apoptosis assay (caspase 3/7 activation)	Hep G2 cells	Measure NP apoptotic effects (caspase 3/7 activation) on liver cells
LLC-PK1 kidney cytotoxicity assay	Porcine proximal tubule cells (LLC-PK1)	Measure NP cytotoxic effects on kidney cells by MTT and LDH release assay
LLC-PK1 kidney apoptosis assay (caspase 3 activation)	LLC-PK1 cells	Measure NP apoptotic effects (caspase 3 activation) on kidney cells
Autophagy
Autophagic dysfunction in LLC-PK1 cells	LLC-PK1 cells	Evaluate NP effects on causing lysosomal dysfunction using lysotracker dye
Qualitative analysis of MAP LC3I to LC3-II conversion by western blot	LLC-PK1 cells	Evaluate NP effects on causing lysosomal dysfunction by immunoblotting

DC: Dendritic cell; MTT: (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NK: Natural killer; NP: Nanoparticle; ROS: Reactive oxygen species.

Sidebar

Executive Summary

The therapeutic potential of siRNAs for a broad range of diseases has already been well demonstrated by bench studies in the past decade. Clinical translation has become the current priority for siRNA research.
Inefficient delivery of siRNAs is often identified as a key obstacle against their clinical translation. Several classes of nanocarriers made from polymers, lipids, inorganic materials or a combination of these have been developed to improve siRNA delivery.
Many 'inert' materials become harmful to the human body at organ, tissue and cell levels once they are downsized to a nanoscale, which results in nanotoxicity.
Nanotoxicity can be caused by the disruption of cell membrane integrity, damage to critical cell organelles, production of reactive oxidative species, lysosomal overload, altered gene expression and stimulation of immunogenic responses.
Nanotoxicity is particularly an issue in siRNA nanocarrier development due to their polycationic charges, complex design and strong interaction with cells and tissues; however, this issue is often overlooked.
Many polymeric and lipid-based siRNA nanocarriers have multiple cationic groups that are associated with significant toxic effects. A chemical engineering approach has been studied to systematically develop less toxic nanomaterials. The use of low molecular weight, hydrolysable, bioreducible, well-established natural materials or inorganic matters are also appealing options.
Several other nanotoxicity reduction strategies include optimization of physical properties of nanocarriers, use of an alternative route of administration, a low-dose approach and the development of safer hybrid nanocarriers.
Currently, there is no well-agreed standard for nanotoxicity evaluation. Researchers have begun to realize that toxicity is an issue and more accurate and clinically relevant methodology is being developed for nanotoxicity assessment.