Biodistribution and excretion of colloidal gold nanoparticles after intravenous injection: Effects of particle size

Abstract

Background:

Inorganic gold nanoparticles (NPs) have a huge potential in targeted drug delivery. Simple preparation and surface modification procedure with their special physicochemical properties of gold NPs attract their use such as tumor targeting and the detection of cancerous cell.

Objective:

Various studies were reported, however, details of biodistribution profile of gold NPs were not enough evaluated. We have studied biodistribution profile of gold NPs having various particle sizes (20, 50 and 100 nm).

Methods:

Gold concentrations in brain, heart, lungs, liver, stomach, pancreas, spleen, kidneys, blood, urine, and feces were measured at 5 minutes, 0.25, 0.5, 1, 2, 3, 6, 12, 18 and 24 hours after administration of gold NPs using inductively coupled plasma atomic emission spectrometry.

Results:

In lungs and brain, especially 20-nm gold NPs were accumulated after 2-3 hours of dose administration, and they were kept for 24 hours, whereas they showed relatively low accumulation in heart, stomach and pancreas. After 12 hours, 3.3–14.4% of the injected gold were observed in fecal matter and urine.

Conclusions:

From this study, the application of gold NPs for targeted delivery to lungs and brain and the excretion route of the gold NPs from the body were suggested

Keywords

Gold nanoparticles biodistribution intravenous mice excretion route

1. Introduction

For the delivery of drug and diagnostic agent, nanotechnology has a huge potential. Recently, much attention has been paid to the nanoparticles, and micro- and nanosized particles have been used as an effective drug delivery device. Many organic carriers are studied in this field, colloidal gold nanoparticles are also investigated. Inorganic gold nanoparticles (NPs) of various size ranges offered several opportunities in targeted drug delivery, imaging and as a diagnosis [1–6]. Simple preparation and surface modification procedure with their special physicochemical properties of gold NPs attract their use in the tumor targeting and the detection of cancerous cells [7,8]. Similar to silver NPs, gold NPs have unique optical properties due to surface plasmon resonance. This property makes gold NPs useful for many applications such as spectroscopy, Raman sensors and bright optical tags for molecular-specific biological imaging [9–11]. Various studies were reported for the use of gold NPs as a carrier for the delivery of drug, DNA, siRNA, gene and as therapeutic application [12–17]. In vivo study reported no toxic effect of gold NPs having 12.5 nm particle size and are mainly trapped in the brain, kidneys, liver, spleen, lungs [18]. In vitro size-dependent cytotoxicity study revealed gold NPs of 1.4 nm (Au₅₅ cluster) had toxicity, while that of 0.8, 1.2, 1.8 nm and 15 nm were relatively safe. The results of cytotoxicity of gold NPs having 1.4 nm particle size were explained by the immobility of DNA which is caused in the major grooves of the DNA. This assumption is supported by the fact that toxicity of the gold NPs reflects a subtle difference of its diameter and by the results of observation that gold NPs of 1.4 nm induces necrosis, whereas gold NPs of 1.2 nm and 1.8 nm cause apoptosis [19,20]. Gold NPs are generally considered as a biocompatible, however, some studies raise the question about the chronic toxicity, in vivo fate and route of excretion. In previous studies, we have reported the synthesis of gold NP having different particle sizes mainly 15, 50, 100, and 200 nm and their biodistributions, and their biodistribution data at 24 hours after administration revealed particle size dependence. The gold NPs with a diameter of 15 nm showed the higher distribution in tissues compared to larger size gold NPs, and mainly accumulated in the liver followed by lungs and kidneys. The Smaller percentage was also observed in spleen, brain, heart, blood and stomach [20]. This result is important to assume in vivo fate and route of excretion of gold NPs, however, more detailed research is required to reveal gold NPs characteristics in vivo [21].

In the present study, we have observed the details of biodistribution profile of gold NPs having three different sizes in mice following intravenous injection to evaluate their in vivo kinetics, and gold amount in fecal matters and urine samples to confirm the excretion route and time of gold NPs.

2. Materials and Methods

2.1. Materials

Hydrogen tetrachloroaurate(III) tetrahydrate ( ${HAuC}_{l 4} ∙ 4 H_{2} O$ , $purity ⩾ 99 %$ ), trisodium citrate dihydrate ( $C_{6} H_{5} {Na}_{3} O_{7} ∙ 2 H_{2} O$ , $purity ⩾ 99 %$ ), polyoxyethylene (20) sorbitan monooleate (polysorbate 80), nitric acid (HNO₃, 60–61%), hydrochloric acid (HCl, 35–37%), gold standard solution (1000 ppm, for atomic absorption spectrochemical analysis) and $d (+)$ -Glucose (C₆H₁₂O₆, purity $y ⩾ 98 %$ ) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

2.2. Preparation of gold NPs

Gold NPs were synthesized by using the method previously described. [21–23]. As shown in Table 1, three different sizes of gold NPs were synthesized from 10 mg/mL of hydrogen tetrachloroaurate(III) tetrahydrate solution and 10 mg/mL of trisodium citrate dihydrate solution, which were prepared by dissolving them in purified water.

Table 1
Experimental parameters of GNPs synthesis

Sample # 10 mg/mL of Hydrogen tetrachloroaurate(III) tetrahydrate solution (mL) Purified water (mL) 10 mg/mL of Trisodium citrate dihydrate solution (mL) Reflux time (min) Centrifugation condition

1 25 160 15 10 40000 rpm, 15 min

2 1 198 0.66 8 8000 rpm, 10 min

3 1 198 0.57 8 5000 rpm, 10 min

Sample #	10 mg/mL of Hydrogen tetrachloroaurate(III) tetrahydrate solution (mL)	Purified water (mL)	10 mg/mL of Trisodium citrate dihydrate solution (mL)	Reflux time (min)	Centrifugation condition
1	25	160	15	10	40000 rpm, 15 min
2	1	198	0.66	8	8000 rpm, 10 min
3	1	198	0.57	8	5000 rpm, 10 min

To synthesize sample #1, 25 mL of 10 mg/mL of hydrogen tetrachloroaurate(III) tetrahydrate solution was added to the 160 mL of purified water, and then subsequently added to three necks round bottom flask equipped with a reflux condenser. The hydrogen tetrachloroaurate(III) tetrahydrate solution was then heated at 100°C in oil bath, and 15 mL of trisodium citrate dihydrate solution was added to the prepared solution. During this procedure, the color change of hydrogen tetrachloroaurate(III) tetrahydrate solution was observed. The solution was refluxed for 10 minutes. After that, it was cooled to room temperature using ice bath. To disperse the synthesized gold NPs, polysorbate 80 was added to the solution at 3 g/L. Finally, it was centrifuged 15 minutes at 4000 rpm and washed with purified water three times. Samples #2 and 3 were synthesized same procedure at conditions shown in Table 1.

2.3. Characterization of gold NPs

Particle size distribution of the samples #1–3 was determined using ELSZ-1000ZS (OTSUKA ELECTRONICS Co., Ltd., Hirakata, Japan) which is one of the dynamic light scattering systems. They were appropriately diluted with purified water and their average particle size was measured at 25°C. Also, the zeta potential of the samples #1–3 was measured at 37°C following resuspended in phosphate buffer (pH 7.4, 0.154 M).

In order to determine contained an amount of gold in the samples #1–3, 200 μL of suspensions of samples #1–3 were added in 800 μL of aqua regias (a mixture of 200 μL of nitric acid and 600 μL of hydrochloric acid) to dissolve gold. Subsequently, 9 mL of purified waters were added to them and vortexed. 1mL of these samples were directly analyzed using ICPE-9000 (Shimadzu Corporation, Kyoto, Japan) which is one of the inductively coupled plasma atomic emission spectrometry. The contained amounts of gold were determined by previously reported method [21,22], by comparing the value with the calibration curve of the gold standard solution.

2.4. Biodistribution study

2.4.1. Animals

Mice (ddY, 7–8 weeks old, male) were housed in stainless steel cages under standard environmental conditions ( $23 \pm 1 ° C$ , $55 \pm 5 %$ humidity and a 12/12 hours light/dark cycle) and maintained with free access to water and a standard laboratory diet (carbohydrates 30%; proteins 22%; lipids 12%; vitamins 3%) ad libitum (Nihon Nosan Kogyo Co., Yokohama, Japan). They were used in accordance with the Guidelines for Animal Experimentation of Tokyo University of Science, which are based on the Guidelines for Animal Experimentation of the Japanese Association for Laboratory Animal Science.

2.4.2. Experimental design

On the day of experiments, mice were housed in metabolic cage 3600M021 (Tecniplast Japan Co., Ltd., Tokyo, Japan) and divided into three groups ( $n = 3$ ). Isotonic suspensions of samples #1–3 were injected to mice, which were made with $d (+)$ -Glucose, via the tail vein at a dose of 7 μL/gram of mice weight with anesthesia. The injections were well tolerated and no adverse effects were observed during the 24 hours observation period. After 5 minutes, 0.25, 0.5, 1, 2, 3, 6, 12, 18, and 24 hours of sample administration, mice were killed by cervical dislocation with anesthesia and blood was collected through inferior vena cava. Blood, urine, feces and all tissues including brain, heart, lungs, liver, stomach, pancreas, spleen and kidneys were collected and weighed. The tissues were briefly washed with normal saline and dried with blotting paper. The isolated tissues were weighed accurately and were stored in a freezer at $- 40 ° C$ until analysis. Blood samples were stored in a refrigerator at 4–6°C.

2.4.3. Quantitative determination of gold from collected tissues

Collected tissues were processed by a wet ashing method. Briefly, tissues in 6 mL nitric acid were heated to 100°C for 30 minutes. Subsequently, 9 mL hydrochloric acid was added and heated 3 hours with being covered with a watch glass. Melted sample solutions were filtered with 200 nm membrane filter, and analyzed using ICPE-9000. The validation of ICP emission spectroscopy methods for the quantitative determination of gold in blood and tissues samples was previously reported by our research group [22].

3. Results and discussion

3.1. Characterization of gold NPs

Synthesis of gold NPs by reduction of reducing hydrogen tetrachloroaurate(III) tetrahydrate with trisodium citrate dihydrate was well correlated with previous data with certain modifications [22]. Trisodium citrate dihydrate concentration imparts a very crucial role for the preparation and control of particle size of gold NPs [24]. To obtain desire particle size of the gold NPs, tetrachloroaurate(III) tetrahydrate concentration and trisodium citrate dihydrate concentration were varied. The data are shown in Table 2. The average particle sizes of samples #1–3 were $19.1 \pm 6.1$ , $54.4 \pm 24.9$ , and $102.7 \pm 45.9 nm$ , respectively. Fig. 1 shows the particle size distribution of them. At a lower concentration of trisodium citrate dihydrate results in an increase in particle size of gold NPs attributed to more gold atoms will aggregate into a nanoparticle [25].

Fig. 1.

Particle size distribution of samples #1–3 ( $n = 3$ ).

Table 2

Particle size, zeta potential and GNPs concentration of samples #1–3 ( $mean \pm SD$ , $n = 3$ )

Sample #	Particle size (nm)	Zeta potential (mV)	Gold concentration of isotonic GNPs suspension (ppm)	Gold injected per gram of mice weight (μg)	Number of particles injected per gram of mice weight
1	19.1 ± 6.1	−40.1	$625 \pm 6.2$	4.38	$7.77 \times 10^{21}$
2	54.4 ± 24.9	−38.2	$640 \pm 6.4$	4.48	$3.44 \times 10^{20}$
3	102.7 ± 45.9	−37.6	$615 \pm 6.1$	4.31	$4.92 \times 10^{19}$

As shown in Table 2 gold NPs showed negative zeta potential. Slightly increase in zeta potential was observed with increase in particle size attributed to the varying citrate ion concentration used for the synthesis of gold NPs. A negative zeta potential suggests good colloidal stability of gold NPs in suspension. Earlier reported study revealed nanoparticles having a zeta potential exceeding $\pm 30 mV$ showed better stability in suspension, as the surface charge prevents aggregation of the particles [26].

Gold contents in gold NPs of different particle sizes were analyzed using ICPE-9000. Table 2 shows the amount of gold injected per gram of mice weight and also a number of gold NPs particles injected per gram of mice weight. The number of gold NPs injected per mice was found to be smaller with an increase in particle size mainly because of particle diameter, as we kept the dose of gold NP constant (about 4.4 μg/g of mice weight).

Fig. 2.

Gold accumulation of samples #1–3 in mice brain, heart, pancreas, and stomach at the various time point of dose administration ( $mean \pm SD$ , $n = 3$ ).

Fig. 3.

Gold accumulation of samples #1–3 in mice kidneys, spleen, lungs, and liver at the various time point of dose administration ( $mean \pm SD$ , $n = 3$ ).

Fig. 4.

The gold amount of samples #1–3 in mice urine, feces, and blood at the various time point of dose administration ( $mean \pm SD$ , $n = 3$ ).

Fig. 5.

Biodistribution of samples #1–3 in mice at various time point of dose administration. (a) 3 hours. (b) 6 hours. (c) 12 hours. (d) 18 hours. (e) 24 hours. ( $mean \pm SD$ , ∗ $P < 0.05$ , $* *$ $P < 0.01$ Tukey’s test, $n = 3$ ).

3.2. Biodistribution study

As shown in Figs. 2–4, samples #1–3 were rapidly cleared from the blood circulation and distributed to the various tissues. Interestingly, sample #1 was not detected in the blood circulation and sample #2 was detected very few after 1 hour, whereas sample #3 were found in circulation after 12 hours. It suggests that relatively large particles have better retention, though their concentration were less than 2% of the injected dose. Similar to previous data, samples #1–3 were found to accumulate mainly in the RES organ, liver and spleen [22]. As shown in Fig. 5, the gold concentration in the spleen was found to decrease with increase in gold NPs sizes after 3 hours post-administration. Then, the significant difference was obtained in this RES organ at 24 hours. This result suggested that the larger gold NPs cleared by the liver, and gold NPs filtered from the liver were cleared by the spleen. Similar trends were confirmed in the lungs and kidneys, and significant differences were also obtained. Previously, PEG-modified gold nanoparticles for gene delivery having particle size 89 nm was reported. The author revealed that when complexes of DNA with PEG-modified gold NPs at a weight ratio of 8.4 (w/w) were intravenously administered to mice, 20% of the injected gold concentration was found in blood at 2 hours, whereas 30% of the gold concentration was detected in liver [27]. This rapid decrease of gold in blood and high accumulation in the liver are similar to our results on gold NPs. We have observed that after 3 hours, the gold NPs were started to accumulate in the lungs and last for 24 hours which could be attributed to the adsorption of proteins on the gold NPs surface may alter the particle size and clear by alveolar macrophage. Sample #1 showed relatively high accumulation in lungs. At heart and pancreas, sample #3 showed relatively high accumulation after 6 hours, and concentrations of samples #1 and 2 were less than 0.2% of the injected dose. Interestingly, the considerable gold concentration was detected in the brain after 3 hours with samples #1–3, which indicated permeation of gold NPs through blood–brain barrier (BBB). Various hypotheses can be drawn for permeation of gold NPs through BBB. The Smaller size of gold NPs may have crossed the BBB through the 20 nm gap between astrocytic end-feet basement membrane and capillary endothelium [28]. Another possibility for the transferred of samples #1–3 attributed to the polysorbate-80 which was used as the stabilizer for the gold NPs. Polysorbate-80-coated nanoparticles were reported to transferred higher amount of drug compared to non-coated nanoparticles by modifying or alter the permeability of BBB by various mechanisms like opening the tight junction and temporary disrupting BBB [29,30]. Also the previous study showed delivery of doxorubicin-loaded nanoparticles functionalized with apolipoprotein A-I to the brain and hypothesized that interaction of apolipoprotein A-I with the scavenger receptor class B type I (SR-BI) located at the BBB [31]. Similarly adsorption of apolipoproteins on gold NPs having a diameter of 20, 50, and 100 nm particle size could have further facilitated transport through BBB. Interestingly, we have found that gold NPs of 20, 50, and 100 nm considerably accumulated in kidneys after 6 hours, and 3.3-14.4% of the injected gold were observed in fecal matter and urine after 12 hours. After 24 hours, gold amounts of three different sizes of gold NPs in urine and feces had no significant difference.

4. Conclusions

In this study, detailed biodistributions of gold NPs have various particle size were studied. They were mainly distributed in liver and spleen at all time points. Smaller gold NPs accumulated in lungs and brain, and they showed low accumulation in heart, stomach, and pancreas. These results suggested that smaller gold NPs have the potential for targeted delivery to lungs and brain. Excretion of gold in feces and urine were also observed, and effects of particle size were not observed. This information will contributed to addressing the questions related to the safety of colloidal gold NPs for chronic administration.

Footnotes

Acknowledgement

This work was supported by Program for Development of Strategic Research Center in Private Universities supported by MEXT (2010-2014).

Conflict of interest

The authors have no conflict of interest to report.

References

Sperling

R.A.

Rivera Gil

Zhang

Zanella

and Parak

W.J.

, Biological applications of gold nanoparticles, Chem. Soc. Rev. 37 (2008), 1896–1908. doi:10.1039/b712170a.

Boisselier

and Astruc

, Gold nanoparticles in nanomedicine: preparations, imaging, diagnostics, therapies and toxicity, Chem. Soc. Rev. 38 (2009), 1759–1782. doi:10.1039/b806051g.

Giljohann

Seferos

Daniel

Massich

Patel

and Mirkin

, Gold Nanoparticles for Biology and Medicine, Angew. Chem. Int. Ed. 49 (2010), 3280–3294. doi:10.1002/anie.200904359.

Shaoo

S.K.

and Labhasetwar

, Nanotech approaches to drug delivery and imaging, Drug Discov. Today 8 (2003), 1112–1120. doi:10.1016/S1359-6446(03)02903-9.

Takeuchi

Fukuda

Kobayashi

and Makino

, Transdermal delivery of estradiol-loaded PLGA nanoparticles using iontophoresis for treatment of osteoporosis, Biomed. Mat. Eng. 27 (2016), 475–483.

Takeuchi

Tomoda

Hamano

and Makino

, Effects of physicochemical properties of poly(lactide-co-glycolide) ondrug release behavior of hydrophobic drug-loaded nanoparticles, Colloids Surf. A: Physicochem. Eng. Aspects 520 (2017), 771–778. doi:10.1016/j.colsurfa.2017.02.054.

Choi

C.H.J.

Alabi

C.A.

Webster

and Davis

M.E.

, Mechanism of active targeting in solid tumors with transferrin-containing gold nanoparticles, Proc. Natl. Avad. Sci. USA 107 (2010), 1235–1240. doi:10.1073/pnas.0914140107.

Medley

C.D.

Smith

J.E.

Tang

Bamrungsap

and Tan

W.H.

, Gold nanoparticle-based colorimetric assay for the direct detection of cancerous cells, Anal. Chem. 80 (2008), 1067–1072. doi:10.1021/ac702037y.

Tian

Z.Q.

Ren

and Wu

D.Y.

, Surface-enhanced Raman scattering: From noble to transition metals and from rough surfaces to ordered nanostructures, J. Phys. Chem. B 106 (2002), 9463–9483. doi:10.1021/jp0257449.

10.

Willets

K.A.

Van Duyne

R.P.

Localized surface plasmon resonance spectroscopy and sensing, Annu. Rev. Phys. Chem. 58 (2007), 267–297. doi:10.1146/annurev.physchem.58.032806.104607.

11.

Jain

P.K.

Huang

El-Sayed

I.H.

and El-Sayed

M.A.

, Review of some interesting surface plasmon resonance-enhanced properties of noble metal nanoparticles and their applications to Biosystems, Plasmonics 2 (2007), 107–118. doi:10.1007/s11468-007-9031-1.

12.

Tsai

C.Y.

Shiau

A.L.

Cheng

P.C.

Shieh

D.B.

Chen

D.H.

Chou

C.H.

Yeh

C.S.

and Wu

C.L.

, A biological strategy for fabrication of Au/EGFP nanoparticle conjugates retaining bioactivity, Nano Lett. 4 (2004), 1209–1212. doi:10.1021/nl049523l.

13.

Rosi

N.L.

Giljohann

D.A.

Thaxton

C.S.

Lytton-Jean

A.K.R.

Han

M.S.

and Mirkin

C.A.

, Oligonucleotide modified gold nanoparticles for intracellular gene regulation, Sci. 312 (2006), 1027–1030. doi:10.1126/science.1125559.

14.

Noh

S.M.

Kim

W.K.

Kim

S.J.

Kim

J.M.

Baek

K.H.

and Oh

Y.K.

, Enhanced cellular delivery and transfection efficiency of plasmid DNA using positively charged biocompatible colloidal gold nanoparticles, Biochim. Biophys. Acta 1770 (2007), 747–752. doi:10.1016/j.bbagen.2007.01.012.

15.

Giljohann

D.A.

Seferos

D.S.

Prigodich

A.E.

Patel

P.C.

and Mirkin

C.A.

, Gene regulation with polyvalent siRNA-nanoparticle conjugates, J. Am. Chem. Soc. 131 (2009), 2072–2073. doi:10.1021/ja808719p.

16.

Pissuwan

Niidome

and Cortie

M.B.

, The forthcoming applications of gold nanoparticles in drug and gene delivery systems, J. Cont. Rel. 149 (2011), 65–71. doi:10.1016/j.jconrel.2009.12.006.

17.

Huang

Jain

P.K.

El-Sayed

I.H.

and El-Sayed

M.A.

, Plasmonic photothermal therapy (PPTT) using gold nanoparticles, Lasers Med. Sci. 23 (2008), 217–228. doi:10.1007/s10103-007-0470-x.

18.

Lasagna-Reeves

Gonzalez-Romero

Barria

M.A.

Olmedo

Clos

Sadagopa Ramanujam

V.M.

Urayama

Vergara

Kogan

M.J.

and Soto

, Bioaccumulation and toxicity of gold nanoparticles after repeated administration in mice, Biochem. Biophys. Res. Com. 393 (2010), 649–655. doi:10.1016/j.bbrc.2010.02.046.

19.

Schmid

, The relevance of shape and size of Au55 clusters, Chem. Soc. Rev. 37 (2008), 1909–1930. doi:10.1039/b713631p.

20.

Pan

Neuss

Leifert

Fischler

Wen

Simon

Schmid

Brandau

and Jahnen-Dechent

, Size-dependent cytotoxicity of gold nanoparticles, Small 3 (2007), 1941–1949. doi:10.1002/smll.200700378.

21.

Sonavane

Tomoda

Sano

Ohshima

Terada

and Makino

, In vitro permeation of gold nanoparticles through rat skin and rat intestine: Effect of particle size, Coll Surf B 65 (2008), 1–10. doi:10.1016/j.colsurfb.2008.02.013.

22.

Sonavane

Tomoda

and Makino

, Biodistribution of colloidal gold nanoparticles after intravenous administration: effect of particle size, Coll. Surf. B 66 (2008), 274–280. doi:10.1016/j.colsurfb.2008.07.004.

23.

Turkevich

Stevenson

and Hillier

, A study of the nucleation and growth processes in the synthesis of colloidal gold, Discuss Faraday Soc 11 (1951), 55–60. doi:10.1039/df9511100055.

24.

Kumar

Gandhi

K.S.

and Kumar

, Modeling of Formation of Gold Nanoparticles by Citrate Method, Ind. Eng. Chem. Res. 46 (2007), 3128–3136. doi:10.1021/ie060672j.

25.

Y.Q.

Liu

S.P.

Kong

and Liu

Z.F.

, A study on the sizes and concentrations of gold nanoparticles by spectra of absorption, resonance Rayleigh scattering and resonance non-linear scattering, Spectrochim. Acta A 61 (2005), 2861–2866. doi:10.1016/j.saa.2004.10.035.

26.

Mohanraj

V.J.

and Chen

, Nanoparticles – a review, Trop. J. Pharm. Res. 5 (2006), 561–573. doi:10.4314/tjpr.v5i1.14634.

27.

Kawano

Yamagata

Takahashi

Niidome

Yamada

Katayama

and Niidome

, Stabilizing of plasmid DNA in vivo by PEG-modified cationic gold nanoparticles and the gene expression assisted with electrical pulses, J. Cont. Rel. 111 (2006), 382–389. doi:10.1016/j.jconrel.2005.12.022.

28.

Hawkins

B.T.

and Davis

T.P.

, The blood-brain barrier/neurovascular unit in health and disease, Pharmacol. Rev. 57 (2005), 173–185. doi:10.1124/pr.57.2.4.

29.

Ramge

Unger

R.E.

Oltrogge

J.B.

Zenker

Begley

Kreuter

and Von Briesen

, Polysorbate-80 coating enhances uptake of polybutylcyanoacrylate (PBCA)-nanoparticles by human and bovine primary brain capillary endothelial cells, Eur. J. Neurosci. 12 (2000), 1931–1940. doi:10.1046/j.1460-9568.2000.00078.x.

30.

Kreuter

, Influence of the surface properties on nanoparticle-mediated transport of drugs to the brain, J Nanosci Nanotechnol 4 (2004), 484–488. doi:10.1166/jnn.2003.077.

31.

Petri

Bootz

Khalansky

Hekmatara

Müller

Uhl

Kreuter

and Gelperina

, Chemotherapy of brain tumour using doxorubicin bound to surfactant-coated poly(butyl cyanoacrylate) nanoparticles: Revisiting the role of surfactants, J. Cont. Rel. 117 (2007), 51–58. doi:10.1016/j.jconrel.2006.10.015.