Quantitative Nanoparticle Tracking

Extracellular Transport of NPs

The effective transport of NPs through extracellular barriers, such as mucus and interstitium, may be critically important in order to achieve the desired therapeutic and/or diagnostic goals. Particle tracking can be an invaluable tool to probe the quantitative transport properties of engineered nanomaterials as they move through these complex barriers. Information gathered from particle-tracking experiments can feedback into the design process to improve NP physicochemical properties and reach design aims.

Mucus is composed of mucin fibers in a low viscosity interstitial fluid, and protects the body against toxic particles and pathogens by steric exclusion and adhesive trapping. Pathogens immobilized in mucus are then cleared rapidly. These same functions, however, also make mucus a potentially critical barrier to the transport of NPs if the target cells are underneath the mucus layer. NPs need to evade mucoadhesive trapping, navigate the lower viscosity pores of the gel and transport through to the other side rapidly before clearance can occur.

The transport behavior of NPs through mucus may be difficult to predict at the nanoscale. This is where particle-tracking technology can prove to be invaluable. In Lai et al., the authors tracked the transport of COOH-PS NPs of various physicochemical properties in mucus to determine which properties allowed the NPs to diffuse fastest through mucus. Surprisingly, they found that 200–500-nm particles are able to move faster in mucus than 100-nm particles.^[28] This study exemplifies this nonintuitive behavior, so particles must be studied carefully using quantitative tools, such as particle tracking. In cystic fibrosis (CF) sputum, 100-, 200- and 500-nm COOH-PS NPs have large variant transport rates, with smaller NPs having few but fast-moving populations.^[29] Furthermore, COOH-PS NPs may be more adhesive to CF sputum than amine-modified particles.

To improve the mucus transport of NPs, the effect of an antiadhesive coating provided by PEGylation was investigated with particle tracking. In Wang et al., the authors looked at different levels of PEGylation of COOH-PS NPs and their ability to speed-up travel through mucus.^[30] Results indicate high coverage provided by the smaller 2-kDa molecular weight PEG is best for this purpose and produces a 1000-fold increase in MSD. In Cu et al., a diffusion model based on Fickian mass transport was used to solve the effective diffusion coefficients of various PEGylated and unmodified NPs in cervical mucus.^[31] NPs of 170 nm and coated with PEG to neutralize surface charge move unhindered in the mucosal gel, suggesting PEGylated vectors can deliver greater drug doses to the underlying epithelium. PEGylation of a NP made from a different material, polysebacic acid, increases MSD two- to ten-fold through mucus.^[32]

Using a different material itself can impact transport rates as sub-200-nm poly(D,L-lactic-coglycolic) acid NP can travel through mucus ten-times faster than similarly sized PS NPs.^[33] The authors suggest that differences in particle surface hydrophilicity and aggregate–mucus interaction may have been responsible for the difference in transport between the two particles. Lai et al. went on to develop different-sized nonadhesive PEGylated COOH-PS NPs, and quantified their transport through cervicovaginal mucus using particle tracking.^[34] Results indicate mucus becomes highly permeable to NPs smaller than 500 nm, which makes small nonadhesive delivery systems ideal for mucosal delivery. In fact, the engineered NPs display greater transport rates than herpes simplex viruses that are even slightly smaller in size.^[35] Remarkably, effective diffusivities of the NPs in cervicovaginal mucus are just slightly lower than in water. In Suk et al., the authors were interested in developing a delivery vector that would be able to penetrate CF mucus.^[36] They discovered that PEGylated COOH-PS NPs up to 200 nm were able to travel through this mucus without sticking.

Delivering NPs to the tumor interstitium is an important goal for many NP-based technologies. Studying this process in a quantitative fashion will unveil ways to improve NP design. Notably, Kawai et al. quantitatively characterized the transport of model drug delivery vectors in vivo through tumor vessels and the interstitium after intravascular injection.^[1] This study uncovered the behavior of different-sized NPs within different interstitial spaces. NP trajectories were constructed from confocal images taken at the perivascular, interstitia and intercellular areas of the xenograft tumor. Experiments show MSD, velocity and diffusion coefficients of the NPs are inversely related to their size. The findings provide guidelines for designing therapeutic NPs that can effectively transport through interstitium to reach tumor cells.

Cite this: Quantitative Nanoparticle Tracking - Medscape - Jun 01, 2011.

Table 1. Intracellular nanoparticle transport studies using particle tracking.
Author (year)	Vector	Vector type	Cargo	Diameter (nm)	Label	Cell	Microscope	Tracking software	Transport parameters	Ref.
Suh et al. (2003)	PEI	Nonviral	Salmon DNA	156	OG514-PEI	COS-7	EF	MetaMorph	MSD, RC	[13]
Suh et al. (2004)	PEI	Nonviral	Salmon DNA	138	OG514-PEI	COS-7	EF	MetaMorph	MSD, D	[14]
de Bruin et al. (2007)	LPEI, EGF-PEG-PEI	Nonviral	pDNA	85–266	Cy5-DNA Cy3-DNA	HuH7	WFF	MATLAB	MSD, D, v	[16]
Bausinger et al. (2006)	PEI	Nonviral	pCMVLuc	85, 165	Cy3-DNA	HuH7	Confocal	Unspecified	MSD, D, v	[15]
Suh et al. (2007)	PEG-COOH-PS	Nonviral	None	100	Proprietary dye	HeLa	EF	MetaMorph	MSD, D, RC	[20]
Lai et al. (2007)	COOH-PS	Nonviral	None	20, 40, 200	Proprietary dye	HeLa, PHUVEC	Confocal	MetaMorph	MSD, D	[22]
Lai et al. (2008)	COOH-PS	Nonviral	None	24, 43	Proprietary dye	HeLa	Confocal	MetaMorph	MSD, D, RC	[21]
Sauer et al. (2009)	MgNP, lipoplex	Nonviral	GFP Nuc pDNA	388	TMR-MgNP, Cy5-DNA	HuH7	WFF	MATLAB	MSD, D, v	[18]
Gopalakrishnan et al. (2006)	Lipid/QD	Nonviral	None	100	Intrinsic	HEK293	Confocal	Unspecified	MSD	[19]
Jin et al. (2008)	SWNT	Nonviral	D(GT)₁₅	2 × 20	Intrinsic	NIH3T3	Fluorescence	ImageJ	MSD	[2]
Neugart et al. (2007)	Nanodiamond	Nonviral	None	50	Intrinsic	HeLa	Confocal	Unspecified	MSD, D	[17]
Lakadamyali et al. (2003)	Influenza X31	Viral	Unspecified	Unspecified	DiD, Cy3, CypHer5	CHO HeLa	DIC, EF	Unspecified	MSD, v	[24]
Babcock et al. (2004)	Influenza X31, vRNP	Viral	Unspecified	30–100	Cy3-vRNP	BS-C-1	DIC, EF	Unspecified	MSD, v	[25]
Suk et al. (2007)	Ad, PEI	Viral	Salmon DNA	143	OG488-PEI GFP-Ad	Rat primary neuron	Confocal	MetaMorph	MSD, D, RC	[26]
Akita et al. (2010)	R8-lip, Ad	Viral	pDNA	189	Rho-lip, TR-Ad	HeLa	WFF	G-track	MSD, D, v	[27]
Seisenberger et al. (2001)	AAV	Viral	Unspecified	20	Cy5-AAV	HeLa	EF	Unspecified	MSD	[23]

AAV: Adeno-associated virus; Ad: Adenovirus; APS: Amine-modified polystyrene; CD: Cyclodextrin; D: Diffusion coefficient; DIC: Differential interference contrast; DiD: 1,1'-dioctadecyl-3,3,3',3'- tetramethylindodicarbocyanine; EF: Epifluorescence; FCS: Fluorescence correlation spectroscopy; GFP: Green fluorescent protein; HSNF: Human skin normal fibroblast; ICS: Spatio–temporal image correlation spectroscopy; LPEI: Linear polyethylenimine; MgNP: Magnetic nanoparticle; MSD: Mean square displacement; OG: Oregon Green; PEG: Poly(ethylene glycol); PEI: Polyethylenimine; PHUVEC: Primary human umbilical vein endothelial cells; PS: Polystyrene; QD: Quantum dot; R8-lip: Octa-arginine-modified liposome; RC: Relative change; Rho: Rhodamine; SWNT: Single-wall nanotube; TMR: Tetramethylrhodamine; TR: Texas Red; v: Velocity; vRNP: Ribonucleoprotein; WFF: Wide-field fluorescence.

Table 2. Extracellular nanoparticle transport studies using particle tracking.
Author (year)	Vector	Diameter (nm)	Label	ECM	Microscope	Tracking software	Transport parameters	Ref.
Lai et al. (2007)	PEG-COOH-PS	100–500	Proprietary dye	CVM	EF	MetaMorph	MSD, D	[28]
Wang et al. (2008)	PEG-COOH-PS	200	AF 647-PS	CVM	EF	MetaMorph	MSD, D	[30]
Tang et al. (2009)	PSA, PSA-PEG, PLGA	100–200	Dox/PSA, PLGA	CVM, CFS	EF	MetaMorph, MATLAB	MSD	[32]
Lai et al. (2009)	PEG-COOH-PS	100, 200, 500, 1000	Proprietary dye	CVM	EF	MetaMorph	MSD	[34]
Lai et al. (2010)	PEG-COOH-PS, HSV-1	100–1000, 180	Proprietary dye-PS AF488-HSV GFP-HSV	CVM	EF	MetaMorph	MSD, D	[35]
Dawson et al. (2003)	COOH-PS APS	100–500	Proprietary dye	CFS	EF, confocal	MetaMorph	MSD, D	[29]
Suk et al. (2009)	COOH-PS	100, 200, 500	Proprietary dye	CFS	EF	MetaMorph	MSD, D	[36]
Dawson et al. (2004)	PLGA-DDAB COOH-PS	<200	Proprietary dye	PGM	Confocal	MetaMorph	MSD, D	[33]
Cu and Saltzman (2009)	PEG-PLGA	170	Coumarin 6	CM	Fluorescence	ImageJ	D	[31]
Kawai et al. (2009)	QD705, fluosphere	20, 40, 100	Intrinsic	Interstitium	EF	Unspecified	MSD, D, v	[1]

APS: Amine-modified polystyrene; CFS: Cystic fibrosis sputum; CM: Cervical mucus; CVM: Cervicovaginal mucus; D: Diffusion coefficient; Dox: Doxorubicin; ECM: Extracellular matrix; EF: Epifluorescence; GFP: Green fluorescent protein; HSV: Herpes simplex virus; MSD: Mean square displacement; PEG: Poly(ethylene glycol); PGM: Pig gastric mucus; PLGA: Poly(d,l-lactic-coglycolic) acid; PS: Polystyrene; PSA: Poly(sebacic acid); QD: Quantum dot; v: Velocity.

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