Uptake and Biodistribution of Nanoparticles – a Review

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Nanotechnology. Author manuscript; bachelor in PMC 2012 Aug 26.

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PMCID: PMC3158492

NIHMSID: NIHMS315771

Magnetic nanoparticle biodistribution following intratumoral administration

A.J. Giustini

¹Dartmouth Medical Schoolhouse and the Thayer School of Technology, 8000 Cummings Hall, Dartmouth Higher, Hanover, NH 03755 USA

R. Ivkov

²Triton BioSystems, Inc., ^† Chelmsford, MA 01824 U.s.

P.J. Hoopes

¹Dartmouth Medical Schoolhouse and the Thayer Schoolhouse of Engineering, 8000 Cummings Hall, Dartmouth College, Hanover, NH 03755 USA

Abstract

Recently, oestrus generated past iron oxide nanoparticles (IONP) stimulated past an alternating magnetic field (AMF) has shown promise in the treatment of cancer. To determine the mechanism of nanoparticle-induced cytotoxicity, the physical clan of the cancer cells and the nanoparticles must be adamant. We have used transmission electron microscopy (TEM) to define the time dependent cellular uptake of intratumorally administered dextran-coated, core-shell configuration IONP having a mean hydrodynamic diameter of 100-130 nm in a murine breast adenocarcinoma cell line (MTG-B) in vivo. Tumors averaging volumes of 115 mm³ were injected with iron oxide nanoparticles. The tumors were and so excised and stock-still for TEM at fourth dimension 0.1 to 120 hours post IONP injection. Intracellular uptake of IONP was 5.0, 48.eight and 91.1% uptake at 1, 2 and four hours post-injection of IONP, respectively. This information is essential for the effective use of IONP hyperthermia in cancer handling.

Keywords: magnetic nanoparticle, tumor, TEM, biodistribution, breast adenocarcinoma

1. Background

It is well known that exposing tumor cells to modestly elevated temperatures sensitizes them to chemotherapy and radiation and, depending on temperature and exposure time, decreases their viability [one] [2]. The offset utilize of magnetic hyperthermia on tumors was reported in 1957, when Gilchrist et al. demonstrated through in vitro experiments that 5 mg of twenty-100 nm bore Fe_twoO_three nanoparticles in lymph nodes (47 mg of Fe₂O₃ per gram of tissue) could produce a temperature rise of fourteen°C in an AMF of 200-240 Oe at 1.two MHz [iii]. Recent advances in nanoparticle technology have allowed for a promising cell-targeted form of therapeutic hyperthermia [iv] [five] [6] [seven].

When accordingly coated, IONP demonstrate fantabulous biocompatibility with limited toxicity [8]. At the same fourth dimension, these nanoparticles can be constructed to have a very high specific absorption rate (SAR), resulting in excellent heating properties and targeting capabilities [9]. Considerable work has been done to elucidate the biologic, therapeutic and materials properties of matrix-configuration iron oxide nanoparticles [x] [11] [12]. This configuration of nanoparticle is typically composed of several, modest (iii-15 nm) iron oxide crystals distributed inside a polymer matrix [viii].

Another configuration of iron oxide nanoparticle, nigh commonly used in AMF-induced hyperthermia, is the core-shell configuration. In this configuration, larger magnetic iron oxide cores (upwards to approximately 50 nm) are coated with a polymer shell [9]. This configuration typically demonstrates greater SAR than the matrix-configuration IONP [ix] [13] [fourteen] and, thus, are well-suited to therapeutic hyperthermia applications. To appointment, few, if any, studies accept demonstrated the biodistribution of core-beat out IONP [xv]. Thus, boosted piece of work is necessary to demonstrate the interaction of core-shell IONP with cells and tissues. In this study, nosotros study on the cellular uptake parameters of dextran-coated, atomic number 26 oxide cadre-beat nanoparticles.

Theoretical models have been published which have shown that currently available nanoparticles of the concentrations used for nanoparticle hyperthermia volition be unable to generate sufficient heating to accomplish global hyperthermia [16]. These models assume homogeneous distributions of nanoparticles inside a sample; our written report suggests that this model is not direct applicably to many biological systems. In addition, inter-particle distance has been shown to be a critical parameter for the generation of estrus [17]; this report, and others, have shown that IONP localize in vacuoles inside cells. In add-on, studies take demonstrated that iron oxide nanoparticles can be used to accomplish global hyperthermia within tissues [6] [17]. Our data suggest that the distribution of nanoparticles inside tumors is not homogeneous. Rather, the nanoparticles aggregate and this aggregation may aid in local heat deposition.

2. Materials and Methods

2.1 Nanoparticles

The nanoparticles utilized in this experiment were dextran-coated, iron oxide (magnetite, Fe_threeO₄)-core BNF® nanoparticles with an average hydrodynamic diameter range of 100-130 nm (MicroMod GmBH, Rostock, Germany). The mean magnetite core bore was approximately 45 nm. The nanoparticle iron concentration was 14.v mg Atomic number 26/ml (33 mg nanoparticle/ml) in deionized water. The synthesis of these nanoparticles was described by Gruettner, et al [9] and a complete description of their physical characteristics has been published [17].

ii.2 Cells

A murine breast adenocarcinoma prison cell line (MTG-B) [18] was cultured in the Alpha modification of Eagle's Minimal Essential Medium (MEM, HyClone Laboratories, Inc.) with 1% penicillin/streptomycin (Pen-Strep, HyClone Laboratories, Inc., Logan, UT, USA), one% L-glutamine (Mediatech, Inc., Manassas, VA), and 10% fetal bovine serum (FBS, Hycolone Laboratories, Inc.) at 37 degrees Celsius in five% CO₂ atmosphere in an incubator (Queue Systems Inc., Parkersburg, VA, The states).

2.iii Brute Tumor Model

This experiment was canonical past Dartmouth's Institutional Animate being Care and Utilise Committee and all animals were treated humanely, in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animate being Care International (AAALAC). To set cells for implantation in mice, the cells were exposed to trypsin (0.25% trypsin in EDTA, HyClone Laboratory, Inc.), stained with trypan blue (Hyclone Laboratories, Inc.), counted using a hemocytometer (Fisher Scientific Inc. Pittsburg, PA, USA), and then re-suspended in (serum-complimentary, L-glutamine-free, Pen-Strep-free) Alpha MEM media at a concentration of 10^seven cells/mL. For each tumor, 100 μL of this solution was injected intradermally into the shaved flanks (one or two tumors per mouse) of female C3H/HE mice (The Jackson Laboratory, Bar Harbor, ME, U.s.), as described in table 1. Three orthogonal tumor measurements were taken with a digital caliper every two days and the tumor volume was determined using the equation for the book of an ellipsoid. When tumors reached volumes of greater than fifty mm³ they were considered of appropriate size for analysis.

Tabular array ane

This table shows the characteristics of each tumor used in the study.

Mouse	Tumor Location	Tumor Volume (mm3)	IONP Target Injection Volume (μL)	IONP Incubation Time
AA3	Correct Flank	54	N/A	Control
AA4	Left Flank	133	46	v Minutes
AA2	Right Flank	104	36	thirty Minutes
AA2	Left Flank	207	71	1 Hour
AAD3	Correct Flank	seventy	24	1 Hour
AAD4	Right Flank	97	33	1 Hour
EMA3	Correct Flank	94	32	2 Hours
EMA3	Left Flank	105	36	2 Hours
EMB1	Left Flank	107	37	2 Hours
EMA2	Left Flank	107	37	3 Hours
EMA4	Left Flank	89	31	iii Hours
EMB2	Correct Flank	84	29	3 Hours
EMB1	Right Flank	101	35	4 Hours
GG4	Right Flank	208	72	iv Hours
AAE2	Left Flank	106	37	iv Hours
AAD2	Right Flank	112	39	5 Hours
AAE1	Right Flank	127	44	5 Hours
AAE3	RIght Flank	145	50	five Hours
AAE2	Right Flank	113	39	6 Hours
AAC2	Right Flank	fourscore	28	6 Hours
AAC3	Right Flank	99	34	6 Hours
FF1	Correct Flank	127	44	9 Hours
FF3	Right Flank	146	50	xiii Hours
HH1	Correct Flank	141	49	eighteen Hours
AB2	Left Flank	105	36	24 Hours
FF4	Right Flank	121	42	5 Days

Nanoparticles (five mg Fe/cm³ tumor) were injected using a xxx-guess needle (0.34 μL of nanoparticle solution per mm³ of tumor). The tip of the needle was avant-garde into the centre of the tumor and the nanoparticle pause injected over the form of xxx seconds. The tip of the needle remained in identify for five minutes post-injection to optimize nanoparticle distribution. Animals were euthanized and the tumors were excised, sectioned, stock-still overnight, and processed for TEM at the pre-adamant post-inject fourth dimension endpoint (table ii).

Tabular array 2

The number of tumors examined at each nanoparticle incubation time betoken.

Incubation Times	Number of Tumors Examined	% of Nanoparticles Intracellular (± Standard Divergence)
Command (no IONP)	i	N/A
Five Minutes	1	0
30 Minutes	i	0
1 60 minutes	3	five.0 (± 4.6)
Two Hours	3	48.8 (± 13.6)
Three Hours	3	70.7 (± 13.5)
Four Hours	3	91.1 (± nine.half-dozen)
V Hours	iii	87.7 (± 9.five)
Six Hours	3	92.9 (± 6.7)
9 Hours	i	100
Thirteen Hours	one	100
Eighteen Hours	i	100
I Mean solar day	1	100
Five Days	1	100

2.four Transmission Electron Microscopy

Tumor tissue samples were fixed in 200 μL of 4% glutaraldehyde (Ted Pella, Inc., Redding, CA, Usa) solution overnight and transferred to 200 μL of 0.one M sodium cacodylate buffer solution (pH 7.4, Ted Pella, Inc.) after three wash cycles with buffer. The samples were prepared for TEM (FEI Company Tecnai F20 FEG TEM operating at 100 kV) at the Dartmouth College Electron Microscopy Facility. Either Fifty.R. White (Polysciences, Inc., Warrington, PA, USA) or Poly/Bed-812 (Polysciences, Inc.) was used as an embedding resin. Samples were stained with 4% osmium tetroxide (Ted Pella, Inc.) and en-bloc stained with 2% uranyl acetate (Ted Pella, Inc.) for one hour, each. Thin sections of 100-110 nm from each tumor sample were cutting using a Leica Ultra-Cut Microtome (Leica Microsystems GmbH, Wetzlar, Germany).

2.v Image Analysis to Quantify Uptake

To quantify nanoparticle uptake, computer code was written using Matlab seven.half dozen.0.324 (The Mathworks, Inc., Natick, MA, USA) Image Processing Toolbox. Ten low-magnification (5000x), randomly-chosen TEM fields from each tumor sample were digitally photographed. This magnification allowed for the assessment of multiple cells and associated extracellular spaces within one field of view. Due to electron density similarities—but morphologic differences— of nuclear chromatin and IONP, nuclei were manually excluded from images using a digital computer input tablet (Wacom Technology Corporation, Vancouver, WA, USA). Nanoparticles were never nowadays within nuclei, so this exclusion did not bear upon quantification accuracy. Artifacts from sample grooming were besides excluded. The images of the cells were and so manually segmented along their plasma membranes in lodge to separately quantify internal and external IONP.

Due to the extreme ballot density of the IONP, we were able to segment each epitome to identify simply IONP in a binary map of the intracellular region of involvement (ROI). In the binary ROI, the nanoparticles were given a value of 0 and all other material a value of 1. This ROI map was inverted and summed to count the number of pixels respective to intracellular IONP. In a similar mode, the extracellular space was analyzed using the same grayscale threshold value as the intracellular IONP. One time the pixels corresponding to nanoparticles were determined, an overlay was created to highlight the pixels determined to exist nanoparticles and manual confirmation was completed. Using this analysis technique, the ratio of internal vs. external nanoparticles was quantified.

For each tumor sample (three tumors per time point) at times ane, ii, 3, four, 5 and six hours, the total number of pixels corresponding to intracellular and extracellular nanoparticles was independently quantified. The percentage of intracellular vs. extracelluar IONP was then determined for each sample.

3. Results

Inside the first 60 minutes mail-injection, 95% of the observed IONP are either associated with the external plasma membrane or within the extracellular space (figure 1). The first indication of significant particle uptake past cells occurs two hours after injection when 48.eight ± 14 % of the nanoparticles are observed within the cells.

Transmission Electron Micrographs of In vivo murine mammary adenocarcinoma cells. a: Tumor cells without IONPs. b: Tumor cells 5 minutes post-obit intra-tumor administration of IONP. The vast majority of IONP are aggregated in the extracellular space (pointer). c: Tumor cells one hour following intra-tumor assistants of the nanoparticles. Larger interstitial aggregates of nanoparticles can exist seen (arrow).

4 hours after IONP injection into the tumor, virtually all (91.1 ± 9.6 %) observed nanoparticles are present within tumor cells. The nanoparticle aggregates have get more than ordered due to nanoparticle segregation within intra-cytoplasmic vesicles. Figure 2 demonstrates the intermediate time point of three hours. Afterward six hours of incubation, nigh no IONP are observed outside the cells (effigy 3). Equally seen at 1 day afterwards injection of nanoparticles into the tumors, the nanoparticles are seemingly exclusively contained inside intracellular intra-cytoplasmic vesicles (figure 3). The quantification of IONP position for time points i through 6 hours is presented in figure 4.

Three hours following intra-tumor injection of IONP into a mouse mammary adenocarcinoma, a large number of INOP are aggregated inside (blackness arrow) and outside (white pointer) tumor cells. a: Although some IONP remain outside of the cells, about IONPs are located within large intra-cytoplasmic vesicles. b: This micrograph demonstrates plasma membrane associated IONP and IONP in the process of trafficking through the membrane and cytoplasmic aggregation.

TEM of a mouse mammary adenocarinoma jail cell demonstrating aggregated IONPs within the cytoplasm (black pointer) and adjacent to the nuclear enveloped (white pointer), 24 hours after administration. All IONPs were either aggregated inside cells or had been eliminated from the tumor extracellular space.

One hr post intratumoral IONP injection (5 mg Fe/cm³ tumor), approximately v% of nanoparticles were found to be intracellular (TEM assessment), whereas 4 hours post injection approximately 90% are intracellular. There is no additional uptake at six hours post injection. Error bars indicate standard divergence of the mean.

4. Discussion

These data suggest that virtually all IONP uptake occurs betwixt one and iv hours subsequently intratumoral injection. These information suggest that there are two time domains for intratumorally delivered IONP hyperthermia. Dennis et al. demonstrated that aggregated IONP result in improved heating characteristics when compared with non-aggregated IONP [14]. Thus, once tumor cells have aggregated IONP intracellularly, more rut will be deposited into the tumor upon AMF activation with the same field strength and frequency, resulting in greater tumor cytotoxicity.

Multiple TEM sections of each tumor sample are examined. The dimension of the individual TEM sections is five mm × five mm, at a thickness of 100 nm. While the total volume of tumors sampled with each TEM is relatively small and is assessed in a two dimensional manner, the use of multiple sections over a small region allows for a reasonably accurate 3-D assessment of an private sample.

These data are the get-go to describe the temporal and spatial human relationship of intratumorally delivered IONP. We demonstrate that nanoparticles are, in fact, heterogeneously distributed on a cellular level within intra-cytoplasmic vesicles. Based on the observations made in this study, at that place does not appear to exist a specific anatomic location of the nanoparticles within cells. Information technology is well known that inter-particle interactions affect the magnetic properties of nanoparticles, which results in aggregated IONP displaying higher heating rates [14]. These improved heating effects may lead to increased cytotoxicity and tumor command and are the focus of electric current studies.

5. Conclusions

We have demonstrated that core-shell magnetic iron oxide nanoparticles of average hydrodynamic bore of approximately 100 nm and coated with dextran and chop-chop internalized (90% within four hours) by tumors cells in vivo (figure 5). Once taken upwards by tumor cells, the nanoparticles are trafficked together into large collections. These results will inform hereafter work using magnetic nanoparticles activated with alternating magnetic fields to treat tumors.

These figures demonstrate IONP movement from the extracellular location (1, left figure) to semi-aggregated intracellular (1) and extracellular (2) locations (heart figure) to entirely intracellular aggregation (2, right figure). The intratumoral mail service-injection fourth dimension is indicated beneath the photographs.

Acknowledgements

This work was supported by NIH NCI grant 1U54CA151662-01 and the Dartmouth Eye of Cancer Nanotechnology Excellence (DCCNE). R. Ivkov was formerly employed by Triton BioSystems (at present Aspen MediSys). The P. J. Hoopes laboratory shared a grant with Triton BioSystems (Award Number TSI-4029-08-78777) and received nanoparticles from that company to complete these studies. Triton BioSystems did not participate in whatever fashion in the production of this manuscript. A.J. Giustini gratefully acknowledges support from the Section of Didactics Graduate Assistantship in Areas of National Demand (GAANN) Fellowship and the Thayer School of Applied science Innovation Fellowship.

The authors thankfully acknowledge Katherine Due south. Connolly and Christopher O. Ogomo for their assistance with TEM imaging and Charles P. Daghlian for fruitful discussions about TEM prototype analysis.

Footnotes

^†now Aspen MediSys, LLC.

PACS: 87.19.xj

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