Hollow Silica Nanoparticles Penetrate the Peripheral Nerve and

Letter pubs.acs.org/NanoLett

Hollow Silica Nanoparticles Penetrate the Peripheral Nerve and Enhance the Nerve Blockade from Tetrodotoxin Qian Liu, Claudia M. Santamaria, Tuo Wei, Chao Zhao, Tianjiao Ji, Tianshe Yang, Andre Shomorony, Bruce Y. Wang, and Daniel S. Kohane* Laboratory for Biomaterials and Drug Delivery, Department of Anesthesiology, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, United States S Supporting Information *

ABSTRACT: The efficacy of tetrodotoxin (TTX), a very potent local anesthetic, is limited by its poor penetration through barriers to axonal surfaces. To address this issue, we encapsulated TTX in hollow silica nanoparticles (TTX-HSN) and injected them at the sciatic nerve in rats. TTX-HSN achieved an increased frequency of successful blocks, prolonged the duration of the block, and decreased the toxicity compared to free TTX. In animals injected with fluorescently labeled HSN, the imaging of frozen sections of nerve demonstrated that HSN could penetrate into nerve and that the penetrating ability of silica nanoparticles was highly size-dependent. These results demonstrated that HSN could deliver TTX into the nerve, enhancing efficacy while improving safety. KEYWORDS: Hollow silica nanoparticles, tetrodotoxin, nerve blockade, penetration

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the particles and their pores (see below) that these are highly porous particles.] A size distribution of 28.2 ± 0.9 nm (Figure 1c) was determined by measuring the diameters of 100 individual HSN from the high-magnification TEM images. Dynamic light scattering (DLS) measurements showed a diameter of 36.7 nm (Figure 1d), which agrees well with the result in Figure 1c. HSN possessed a negatively charged surface with a zeta potential of −9.8 mV, suggesting its possibility for loading cation TTX. Below, all silica nanoparticles are described with a subscript denoting their approximate diameter in nanometers, and hollow particles have the prefix “H” (e.g., HSN30). The nitrogen adsorption isotherm curve of HSN30 (Figure S1) showed a type IV adsorption−desorption isotherm,8 which is typical of porous materials. The pore volume was calculated to be 1.43 cm3/g (by the software of the V-sorb 2800 surface area and porosimetry analyzer) and the Brunauer−Emmett−Teller (BET) surface area was 462 m2/g (BET is the most common theory for determining the surface area of powders and porous materials.9). These values suggested that HSN30 were highly porous and could be suitable as carrier for drug delivery.7 After loading with TTX, the pore volume and surface area decreased to 1.11 cm3/g and 355 m2/g (Figure S2), respectively, demonstrating that the TTX was adsorbed inside the pores of the HSNs. The HSN30 had a calculated pore size distribution centered at 13.4 nm (Figure S3).10 There was no significant aggregation of HSN30 after loading with TTX (Figure S4). Loading Efficiency and TTX Release Profile of TTX-HSN30. TTX was loaded by mixing HSN30 and TTX in aqueous solution.

etrodotoxin (TTX) is a very potent local anesthetic that acts by blocking site 1 on the voltage-gated sodium channel, on the axonal surface. Tetrodotoxinand other site 1 sodium channel blockers (S1SCBs), such as the saxitoxinshave minimal local toxicity as well as decreased cardiac and central nervous system toxicity.1 The effectiveness of S1SCBs is limited by relatively poor penetration through various tissue barriers to their site of action; this difficulty is probably due to their hydrophilicity and charge. The high concentrations of S1SCBs required to overcome those barriers and achieve useful degrees and durations of nerve block can entail considerable systemic toxicity.2 Efforts to overcome the barriers have included disrupting them by osmotic shock3 or permeabilizing them with chemical permeation enhancers.4 Another approach has been to use sustained release systems, extending the period during which the nerve is exposed to S1SCBs, and maintaining a high local concentration.5 Here we have hypothesized that nanoencapsulation could enhance S1SCB penetration into the nerve and provide an extended duration of nerve block. We have used hollow silica nanoparticles to deliver TTX (TTX-HSN) as a model S1SCB due to its commercial availability. We selected HSN to deliver TTX because their hollow structure could facilitate loading with TTX. Loading could also be assisted by the negative charge of silica (its isoelectric point is about 2 (ref 6)) while TTX is cationic. The sustained release of TTX would prolong the effect, while reducing systemic toxicity. Results and Discussion. HSN Formulation. HSN were prepared as reported (see Supporting Information).7 Transmission electron microscopy (TEM; Figures 1a and b) of HSN confirmed that they were hollow spheres with uniform particle size. [We note, however, that it is possible given the size of © XXXX American Chemical Society

Received: June 9, 2017 Revised: September 18, 2017

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DOI: 10.1021/acs.nanolett.7b02461 Nano Lett. XXXX, XXX, XXX−XXX

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Figure 1. Characterizations of HSN30. (a, b) TEM images of HSN30 at (a) low and (b) high magnification. (c) Representative (of >10) histogram of the size distribution of HSN30 measured by TEM. (d) Representative (of >10) size intensity-weighted distribution of HSN30 as determined by dynamic light scattering measurements.

in PC12 cells (a pheochromocytoma cell line frequently used in neurotoxicity studies). TTX-HSN30 also did not cause any loss of cell viability for up to 4 days. These results suggested that TTX-HSN30 could be safe for the following animal experiments. Nerve Blockade with TTX and TTX-HSN30. Rats (4 in each group) were injected at the left sciatic nerve with 0.3 mL of water containing 0−6 μg of TTX, either free or in TTX-HSN30 (0−60 mg/mL of HSN30). They then underwent neurobehavioral testing to determine the duration of functional deficits in both hindpaws. The duration of deficits on the injected side reflected the duration of the nerve block. Deficits on the uninjected side (right; contralateral) reflected systemic TTX distribution. Free TTX showed a concentration-dependent increase in the median duration of nerve block (Figure 3a) and frequency of successful nerve blockade (Figure 3b; see Methods for the definition of successful nerve block). Nerve block from 4 μg of free TTX had a median duration of 79.5 min with 80% successful blocks (Figure 3b) and 30% of animals had contralateral deficits (Figure 3c). Six μg of free TTX caused contralateral deficits in all animals and was uniformly fatal. The nerve block duration was significantly prolonged with the TTX-HSN30 (Figure 3a). Sensory nerve blockade with 4 μg of TTX in TTX-HSN30 lasted 274 min (p < 0.005 compared to free TTX); with 6 μg of TTX, it lasted 362 min, and no animals died or had contralateral deficits. In this animal model it is not possible, due to limiting toxicity, to achieve such long nerve blocks with TTX in the absence of sustained release,5a chemical permeation enhancers,4 or drugs that enhance the effect of S1SCBs.11 TTX-HSN30 resulted in a much higher rate of successful nerve blockade than did free TTX: 100% blockade was observed even at a very low dose of TTX (e.g., 1 μg, Figure 3b). This increase in the success rate is not the norm for encapsulated TTX5a but was

Figure 2. Cumulative release of TTX from TTX-HSN30 in vitro. Data are the means with SDs (n = 4).

The mixture was stirred at room temperature for 48 h. The obtained TTX-HSN30 solution was diluted and used for subsequent studies without removing the free TTX. To measure the loading efficiency of TTX, the obtained TTX-HSN30 solution was washed with water three times; the supernatant after centrifugation (12 000 rmp, 20 min) was collected, and the free TTX was measured by enzyme-linked immunosorbent assay (ELISA). The loaded TTX was calculated to be the total amount of TTX added to the HSN30 minus the free TTX. The loading efficiency of TTX in HSN30 was 49.0 ± 2.0%. To assess the potential of these TTX-HSN30 to provide sustained nerve blockade, we performed release kinetic studies at 37 °C in PBS (Figure 2). The TTX-HSN30 significantly increased the duration of TTX release from the system (a dialysis device with a 20 000 MW cutoff), compared with free TTX (e.g., ∼90% for free TTX vs ∼50% for TTX-HSN30 at 6 h, p < 0.005). Cytotoxicity. C2C12 cells (myotube cell line used to assess myotoxicity) were exposed to TTX-HSN30 for up to 4 days (Figure S5). TTX-HSN30 did not reduce cell survival at any duration of exposure tested. Similar studies were performed B

DOI: 10.1021/acs.nanolett.7b02461 Nano Lett. XXXX, XXX, XXX−XXX

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Figure 3. Sciatic nerve blockade with free TTX and TTX-HSN30. Effect of TTX dose on (a) the median duration of sensory nerve blocks, (b) the frequency of successful blocks, (c) the frequency of nerve block in the uninjected (contralateral) extremity, and (d) of death. Data in panel a are medians with interquartile ranges. n = 4 per experimental group.

similar to the effect of chemical permeation enhancers on TTX nerve block.4 Encapsulation in TTX-HSN30 decreased the incidence of systemic toxicity (Figure 3c and d). There was no evidence of systemic toxicity (contralateral sensory deficits or mortality) at any dose ≤6 μg of TTX in TTX-HSN30. This enhanced safety is attributable to the sustained release function from the HSN30. Silica Nanoparticle Distribution. The resemblance of some characteristics of nerve block from TTX-HSN30 to those from TTX with chemical permeation enhancers led us to investigate the possibility that the HSN30 were enhancing TTX flux into nerve. To evaluate that possibility, we synthesized HSN30 to which fluorescein isothiocyanate (a fluorescent dye with an excitation wavelength of 488 nm and emission wavelength of 519) nm was covalently conjugated (see Methods in the SI and Figure S6a) so that the dye would be associated with the particles and not able to diffuse independently; the particles are denoted FITC-HSN30. The diameter of FITC-HSN30 was ∼28 nm (Figure S6b), similar to that of HSN30. The absorbance and fluorescence emission spectra of FITC-HSN30 had peaks at 495 and 520 nm, respectively (Figure S6c). Fluorescent imaging was used to track the location of FITC-HSN30 in tissue. 300 μL (30 mg/mL) of FITC-HSN30 in water was injected at the sciatic nerve. Four hours later, animals were euthanized, and the sciatic nerve was exposed. FITC-HSN30 were identified as a faintly light yellow material around the nerve (Figure S7a). The nerve and surrounding tissue were then harvested. Under irradiation of a 365 nm UV lamp, green fluorescence was observed from sciatic nerve and adjacent muscles in the injected leg (Figure S7b) but not in the untreated leg. Frozen sections of the tissues were produced, and fluorescent images taken. In the animals injected with FITCHSN30, FITC fluorescence was observed in the nerve (Figure 4a and 4b), whereas no FITC fluorescence was observed in the nerve in animals injected with the same dose of free FITC (Figure 4c) or in untreated legs (Figure 4d). Quantitative analysis showed the fluorescence signal penetrated deep into the nerve in animals injected with FITC-HSN30 (Figure 4e

Table 1. Myotoxicity and Inflammation from Free TTX and TTX-HSN30a myotoxicity score free TTX TTX-HSN30 P value

inflammation score

Day 4

Day 14

Day 4

Day 14

0 (0−1.0) 0 (0−0) 0.45

0.5 (0−1.0) 0 (0−0) 0.18

0.5 (0−1.0) 0.5 (0−1.0) 1.00

0.5 (0−1.0) 0 (0−1.0) 0.60

a

The range of scores is 0-4 for inflammation and 0-6 for myotoxicity. P values were determined by Mann-Whitney U test.

and Figure S8), while it remained at the nerve perimeter in animals injected with free FITC. The total fluorescence intensity inside the nerve perimeter was much higher in animals injected with FITC-HSN30 than in those injected with free FITC (Figure 4f, p < 0.005). These results demonstrated that HSN30 can cross the perineurial barrier and penetrate into the nerve. To evaluate the influence of particle size on penetration into nerve, we prepared FITC-labeled silica nanoparticles 9.8 ± 0.5 nm (FITC-SN10; Figure 5a) and 70.0 ± 6.5 nm (FITC-SN70; Figure 5b) in diameter as described.12 They were injected at the sciatic nerve and were harvested at 4 h and processed as were the FITC-HSN30. FITC-SN10 dispersed throughout the nerve (Figure 5c−d), while FITC-SN70 were all located outside the nerve (Figure 5e). Quantitative analysis also confirmed this difference in distribution (Figure 4e). At a normalized distance of 0.1 (one tenth of the diameter of the nerve), the fluorescent intensity in the FITC-SN10 and FITC-HSN30 groups were 72 ± 4.3% and 21 ± 4.0% of the fluorescent intensity at the surface of the nerve, respectively, while that of the FITC-SN70 was only 4 ± 0.5% (both p < 0.005, compared to FITC-SN10 and FITC-HSN30), similar to that obtained with free FITC (p = 0.48). These result demonstrated that the nerve penetrating ability of silica nanoparticles was highly sizedependent. Tissue Reaction. Animals injected with TTX and TTX-HSN30 were euthanized 4 and 14 days after injection (n = 4 at each time C

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Figure 4. Representative fluorescence images of sciatic nerves and surrounding tissues 4 h after injection of (a and b) FITC-HSN30, (c) free FITC, and (d) noninjected leg. Blue, cell nucleus; green, FITC-HSN30 or free FITC. The red dotted line indicates the nerve perimeter. The scale bars are 200 μm. n = 4 per experimental group. (e) Relationship between normalized mean fluorescence intensity (a metric of fluorescence) and normalized distance from the surface of the nerve in animals injected with FITC-SN10, FITC-HSN30, FITC-SN70, or free fluorescein, n = 4. Tissues were harvested 4 h after injection. The method of determining the distance between a given fluorescent area and the surface of the nerves is shown in Supporting Information. The fluorescence intensity at the nerve surface (periphery) was set to 1 and all values normalized to that. The diameter of nerve was set to 1 and all distances from the nerve surface normalized to that. (f) Mean fluorescence ± SD in nerve after treatment with FITC-HSN30 or free FITC, *p < 0.005; n = 4.

Since H&E staining is relatively insensitive for identifying nerve injury, we obtained toluidine blue-stained Epon-embedded sections of the sciatic nerve in animals injected with TTX-HSN30. Nerves from those animals were normal in appearance (Figure 6e−f). Conclusion. In peripheral nerve blockade, the various particulate and other drug delivery systems that have been used to prolong the duration of local anesthetic effect4b are generally thought of as being essentially depot systems that release local anesthetics in the immediate vicinity of the nerve. In that view, the rationale for using nanomaterials for nerve block is not particularly strong,14 since in general larger particles will

point), and the sciatic nerve and surrounding tissues were harvested, sectioned for histology, and stained with hematoxylin− eosin (H&E). These time points are useful in that they can capture acute and chronic inflammatory responses to injected materials. TTX-HSN30 were not observed on gross dissection (Figure S9). Microscopic examination revealed very mild myotoxicity and inflammation 4 and 14 days after injection in animals injected with free TTX and TTX-HSN30 samples (Figure 6a−d). The myotoxicity and inflammation were quantified using a scoring system (Table 1).13 There was no statistically significant difference between the scores for TTX-HSN30 and free TTX. D

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Figure 5. Characterization and performance of FITC-SN10 and FITC-SN70. (a) TEM image of FITC-SN10, (b) TEM image of FITC-SN70. (c−e) Representative fluorescence images of sciatic nerves and surrounding tissues 4 h after injection of (c, d) FITC-SN10 and (e) FITC-SN70. Blue, cell nucleus; green, FITC-SN10 or FITC-SN70. The red dotted line indicates the nerve perimeter. The scale bars are 200 μm. n = 4 per experimental group.

Figure 6. Histology of rat tissues injected with free TTX and TTX-HSN30. (a−d) Representative H&E stained sections of muscles at the site of injection 4 and 14 days after injection. The left scale bar is 200 μm; the right is 50 μm. (e−g) Representative toluidine blue-stained sections of sciatic nerves from animals (e) without and (f and g) with injection of TTX-HSN30. (f) Harvested 4 days after injection and (g) 14 days after injection. (e−f) The scale bars are 100 μm. All animals were injected with 4 μg of TTX, free or in TTX-HSN30 formulation. n = 4 per experimental group.

nanoparticles was highly size-dependent. The sustained release properties of HSNs30 also contributed to the extension of nerve block and enhanced safety by slowing release. This ability to penetrate peripheral nerve could be useful in delivering a range of therapeutics, including combinations of drugs that enhance the activity of local anesthetics.4b

encapsulate more drug, have a slower release, and will be less likely to degrade or leave the site of administration.15 Here we have demonstrated that 28 nm HSN30 containing TTX can penetrate into the nerve. This penetration likely contributed to the increase in the number of successful nerve blocks as well as the prolongation of nerve block. The penetrating ability of silica E

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(9) Gelb, L. D.; Gubbins, K. E. Characterization of Porous Glasses: Simulation Models, Adsorption Isotherms, and the Brunauer− Emmett−Teller Analysis Method. Langmuir 1998, 14 (8), 2097−2111. (10) Grudzien, R. M.; Grabicka, B. E.; Jaroniec, M. Effective method for removal of polymeric template from SBA-16 silica combining extraction and temperature-controlled calcination. J. Mater. Chem. 2006, 16 (9), 819−823. (11) Adams, H. J.; Blair, M. R.; Takman, B. H. The Local Anesthetic Activity of Tetrodotoxin Alone and in Combination With Vasoconstrictors and Local Anesthetics. Anesth. Analg. 1976, 55 (4), 568− 573. (12) (a) Huo, Q. S.; Liu, J.; Wang, L. Q.; Jiang, Y. B.; Lambert, T. N.; Fang, E. A New Class of Silica Cross-Linked Micellar Core-Shell Nanoparticles. J. Am. Chem. Soc. 2006, 128, 6447−7453. (b) Yu, M. H.; Zhou, L.; Zhang, J.; Yuan, P.; Thorn, P.; Gu, W. Y.; Yu, C. Z. A simple approach to prepare monodisperse mesoporous silica nanospheres with adjustable sizes. J. Colloid Interface Sci. 2012, 376, 67−75. (13) McAlvin, J. B.; Padera, R. F.; Shankarappa, S. A.; Reznor, G.; Kwon, A. H.; Chiang, H. H.; Yang, J.; Kohane, D. S. Multivesicular liposomal bupivacaine at the sciatic nerve. Biomaterials 2014, 35 (15), 4557−4564. (14) Santamaria, C. M.; Woodruff, A.; Yang, R.; Kohane, D. S. Drug delivery systems for prolonged duration local anesthesia. Mater. Today 2017, 20 (1), 22−31. (15) Kohane, D. S. Microparticles and nanoparticles for drug delivery. Biotechnol. Bioeng. 2007, 96 (2), 203−209.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.7b02461. Experimental details, N2 adsorption−desorption isotherm, pore size distribution curve of HSN30, TEM image of TTXHSN30, cytotoxicity of TTX-HSN30, charaterization of FITC-HSN30, photographs of sciatic nerve, and measurement of distribution of fluoresecence across the nerve (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Qian Liu: 0000-0002-7498-1425 Tianshe Yang: 0000-0003-0411-7794 Daniel S. Kohane: 0000-0001-5369-5932 Author Contributions

Q.L. and C.S. contributed equally to this work. Q.L. and D.S.K. designed the project. Q.L., C.S., T.W., C.Z., T.J., T.Y., A.S., and B.W. performed the experiments. Q.L., C.S., T.W., and D.S.K. analyzed and interpreted the data. Q.L. and D.S.K. wrote the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by NIH Grant GM073626 (to D.S.K.). We thank the Harvard Medical School EM Facility for technical support.

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REFERENCES

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