Targeted Drug Delivery of Transferrin-Conjugated Mesoporous Silica Nanoparticles

DOI 10.17480/psk.2017.61.5.241

Targeted Drug Delivery of Transferrin-Conjugated Mesoporous Silica Nanoparticles

Muhyun Jang and Injoon Oh

College of Pharmacy, Chonnam National University, Gwangju 61186, Korea (Received August 28, 2017; Revised October 10, 2017; Accepted October 13, 2017)

Abstract — The mesoporous silica nanoparticle (MSN) is a promising carrier as a drug-delivery system. And transferrin has been known as a ligand which binds to a receptor expressed on the surface of most proliferating cells such as tumor cells. In order to further improve the tumor delivery of MSN carrier, transferrin- conjugated MSNs were synthesized and evaluated as a targeting carrier. Fourier transform near infrared data revealed a conjugation of transferrin with MSN, while zeta potential and transmission electron microscopy results showed the nano-characteristics of the MSN in detail. To con- firm the optimal conditions for drug encapsulation onto the MSN, two types of dyes (hydrophilic rhodamine B and hydro- phobic 5,6-carboxyfluorescein) were loaded. Then, the differences in loading efficiencies and release properties of these dye- loaded MSNs were tested. The cellular uptake of dye-loaded MSNs was greater than dye solution. More importantly, the cellular uptake of transferrin modified MSNs was enhanced in the transferrin receptor-expressing Jurkat cells but not in nor- mal cells. Therefore, these results suggest that transferrin-modified MSN could be applied for therapeutic purposes as a drug-delivery system for targeting specific cancer cells.

Keywords Mesoporous silica nanoparticles, transferrin, targeted delivery, drug release, conjugation

Cancer is one of the most life threatening diseases that afflicts individuals all over the world. At present, various kinds of chemotherapeutic drugs have been developed. However, in many clinical cases, they often induce toxicity in normal tis- sues (such as loss of hair, changes in taste, and constant fatigue even under optimal conditions) due to their nonspecific actions. To overcome these side effects, there has been a lot of attention directed towards targeting the drug precisely and effi- ciently to the site of the cancer cell. In this study, the main strategy was to design a target-specific drug-delivery system that can transport an effective dose of the drug molecules to the precise cells and tissues.

The mesoporous silica nanoparticle (MSN) is a silica-based porous carrier that offers benefits such as a tunable particle size, stable and rigid framework, uniform pore size, high sur- face area and large pore volumes. Many studies have shown

that the MSN is biocompatible at the effective dose, and is capable of reducing the toxicity of anticancer drugs.

Nota- bly, MSN has an inner and an outer surface. This specialty allows the selective functionalization of the inner and outer surfaces of the MSN with different functional molecules like proteins or DNA.

MSN-based drug-delivery systems have also been reported to have the ability to bypass multidrug resistance mechanisms such as the P-glycoprotein efflux pump.

There have been several strategies taken to deliver chemo- therapeutic drugs to cancer cells more precisely. These include delivery approaches that avoids the reticuloendothelial sys- tem, utilizes the enhanced permeability and retention (EPR) effects, and are tumor-specific targeted.

Although EPR effects in themselves enable targeted delivery of MSN, devising outer surface functionalization of MSN-based particles with receptor- specific ligands (for example folic acid or IL-13) would further enhance the platform’s therapeutic profile by actively target- ing the pathology and simultaneously lessening the collateral damage.

Here, we used transferrin (Tf) protein as the tar- geting agent. The transferrin receptor (TfR) functions in the cellular uptake of iron through its interaction with Tf. This

#

Corresponding Author Injoon Oh

College of pharmacy, Chonnam National University, Gwangju 61186, Korea

Tel.: 062-530-2927 Fax.: 062-530-2949 E-mail: ijoh@jnu.ac.kr

Short Report

receptor is an attractive molecule for targeted delivery of anti- cancer drugs as it is upregulated on the surface of many types of cancers and is efficiently internalized.

Jurkat cells have increased numbers of transferrin receptors relative to other cell types (from 150-200%).

In this study, Tf-modified MSN (MSN-Tf) was exploited as a novel vehicle for the delivery of chemotherapeutic agents. To test our syn- thesized MSN-Tf, we loaded dyes instead of drugs onto the MSN-based particles as they permit a simple means of obser- vation of delivery, and provide measurable properties com- pared to any anticancer drugs. Two different dyes were carefully selected: rhodamine B (RhB, red) and 5,6-carboxyflu- orescein (CF, green). Each of these dyes were chosen because of their hydrophilic and hydrophobic properties, respectively.

To characterize the MSN-based particles, the size and zeta potentials of these particles were first measured. Further, field- emission transmission electron microscope (FE-TEM) and Fourier transform near infrared spectrometer (FT-NIR) stud- ies were completed to confirm whether the MSN and its deriv- atives were well synthesized and functionalized. Then, the drug contents and their release properties were calculated to determine which property (hydrophilic or hydrophobic) pro- vided a better option for drug encapsulation of MSN. To observe the targeting effects of MSN-Tf, two cell lines were prepared. Human embryonic kidney cells 293 (HEK 293) cells have scarce TfR present, and were used as control cells in this study.

Compared to HEK 293 cells, Jurkat clone E6-1 cells have upregulated TfR on the surface of their cell membranes.

Subsequent cellular uptake tests indicated which cells are more suitable for MSN-based delivery, and demonstrated the targeting effects of MSN-Tf.

Experimental Methods

Materials

Hexadecyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), (3-aminopropyl) triethoxysilane (APTES), sodium cyanoborohydride, sodium m-periodate, holo-transferrin human, 5,6-carboxyfluorescein, and rhodamine B were pur- chased from Sigma-Aldrich, Korea. Dulbecco's modified eagle's medium (DMEM), RPMI 1640, trypsin-EDTA, dulbecco's phos- phate-buffered saline (DPBS), penicillin-streptomycin solutions, and fetal bovine serum (FBS) were purchased from Welgene, Korea. All materials were analytical grade and used without any

further purification.

Preparation of transferrin-conjugated MSN

Aminated mesoporous silica nanoparticle was synthesized using methods described by Suteewong et al.

Briefly, NH

OH (2.94 ml), EtOAc (0.96 ml), and CTAB (60 mg) were sequentially added to 150 ml of distilled water under vigorous stirring conditions. Then, a mixture of APTES and TEOS (0.54 ml) was added to the sample and stirred overnight at room temperature. Next, the MSN products were collected by centrifugation and redispersed in a mixture of methanol and NH

NO

, and stirred for 1h at 70

C to remove any surfactants from its pores.

The nanoparticle products were washed with ethanol and finally lyophilized.

Synthesis of transferrin-conjugated MSN was performed as described in our previous work with some minor modifica- tions.

Transferrin (5 mg) in sodium acetate buffer was mixed in cold sodium m-periodate and stirred for 90min. Then, 100 mg of MSN and sodium cyanoborohydride were added and stirred under dark conditions. The reaction mixture was incubated for 19 hours at room temperature. Next, then nanoparticles were washed with PBS buffer and finally lyophilized.

The transferrin-conjugated MSN was directly analyzed by FT-NIR (Spectrum 400, Perkin Elmer, USA).

Characterization of MSN

Both particle size and the zeta potential of the MSN were determined by an electrophoretic light scattering analyzer (ELS 8000, Otsuka Electronics, Japan). Analysis was run in triplicates after dispersion in distilled water. The structural morphology of MSN was observed by field emission transmission electron microscope (FE-TEM) (JEM-2100F, JEOL, Japan) operated at 200 kV.

Drug loading and loading efficiency of MSN

The drug was loaded by soaking the MSN in a concentrated drug solution and stirred for 24h to achieve a stable drug- loaded nanoparticle. Then, the nanoparticles were centrifuged and lyophilized.

To determine the drug content and loading efficiency of the MSN, the samples were centrifuged after overnight stirring at 17000 rpm; then, the supernatant was removed and analyzed.

The concentration of RhB was measured by UV spectrophotom-

eter (UV-1601, Shimadzu, Japan) at 554 nm, and CF was mea-

sured by spectrofluorometer (FP-6200, Jasco, Japan) at an excitation wavelength of 492 nm and an emission wavelength of 517 nm.

Release of RhB and CF in vitro

Evaluation of in vitro drug release was carried out using the dialysis bag diffusion technique in 10 mL of PBS (pH 4.0 and 7.5) at 37

C, with a shaking rate of 100 rpm. At predeter- mined time intervals, the entire medium was collected and replaced with an equal volume of fresh PBS to maintain sink conditions. The amount of released RhB from MSN was mea- sured by UV spectrophotometer at 554 nm of wavelength. CF was measured by spectrofluorometer at an excitation wave- length of 492 nm and an emission wavelength of 517 nm.

Cell culture and cellular uptake of MSN

HEK 293 cells were obtained from the Laboratory of Toxicol- ogy (Chosun University, Korea), and Jurkat cells were obtained from the Korean cell line bank (Cancer research institute, Seoul National University, Korea). These were maintained in DMEM or RPMI 1640 media supplemented with 10% FBS and 1% pen- icillin, at 37

C in 5% carbon dioxide atmosphere. The media was changed every two or three days, and the cells were passaged by trypsinization before confluence.

HEK 293 cells and Jurkat cells were seeded in 12-well plates (Corning-Coaster, USA), and incubated for 72 h. Next, the cell wells were washed once with DPBS (pH 7.4). Then, the MSN particles were added to each cell well at a concentra-

tion of 50 µg/ml and incubated for 3h. The wells were rinsed once carefully with PBS (pH 7.4), and fluorescence analysis was performed by FACS (FC500, Beckman Coulter, USA).

Results and Discussion

Characterization of transferrin-MSN conjugate

Transferrin conjugates of MSN were synthesized by the perio- diate oxidation method. Synthesis of MSN-Tf was confirmed by FT-NIR spectroscopy. FT-NIR spectra of MSN, free Tf and MSN-Tf are shown in Figure 1. Compared to MSN alone (Fig- ure 1A), new peaks were observed with MSN-Tf at 1541 cm

(NH bending), 1638 cm

(C=N bending), and broadly around 2960 cm

(Figure 1C). These peaks could be attributed to the stretching and bending vibrations of Tf (Figure 1B). These results demonstrate that the aldehyde group of Tf was bound to the amine group of the MSN.

Particle size and zeta potential of MSNs

The particle sizes and zeta potentials of MSN, MSN-Tf, RhB/MSN, RhB/MSN-Tf, CF/MSN and CF/MSN-Tf are shown in Table 1. While all MSN-based particles have an average par- ticle size of 200 −300 nm, Tf modified MSNs or dye-loaded MSNs are slightly larger than unmodified MSNs. As it is widely known, the pore cutoff size of porous blood vessels in a majority of tumors ranges between 380 −780 nm. Accordingly, the range for an EPR effect should be similar. This suggests that MSN-based particles can benefit from the EPR effect as

Fig. 1 − FT-NIR spectra of MSN (A), transferrin (B), MSN-Tf (C). Samples were recorded at a frequency range of 4000-380 cm

.

well as potentially exploit passive targeting with the appropri- ate particle size.

The zeta potential of MSN (25.65 mV) is slightly higher than that of Tf-modified or dye-loaded MSN- based particles. Modifications to Tf affects the net charge of the particles; and accordingly, the loss of the amine group causes the negatively charged transferrin to bind to the sur- face of the MSN. When each dye was loaded onto the MSN, the dyes located near the surface of the MSN had some mild effects on the net charges of the particles. Generally, the less negatively charged, or conversely more positively charged, particles tend to internalize more efficiently by nonphagocytic cells and are less inhibited by the immune system. Addition- ally, zeta potentials of 10-25 mV are sufficient to ensure sur- vival of the particles in vivo as well as uptake by cells.

Morphologies of MSN and MSN-Tf

One of the advantages of MSN as a basic carrier is its meso- pores, which can be efficiently loaded with a large number of hydrophobic small molecules, thereby eliminating the use of tissue-toxic solvents.

The mesopores displayed an aver- age pore diameter of around 1-3 nm, which could be clearly observed, as shown in Figure 2A. Both MSN and MSN-Tf par- ticles displayed a spherical architecture and measured around 200 nm in size as determined by FE-TEM (Figures 2A and 2B). Of note, the mesopores were not that clearly visible (as shown in Figure 2B), which may be attributable to the surface- shielding effect of the modifications of the Tf. However, in fur- ther tests, MSN-Tf showed similar loading and release proper- ties as unmodified MSN, which implies that the mesopores were still present on the surface of MSN-Tf.

Determination of drug contents and loading efficiency The drug contents and loading efficiencies of dye-loaded MSNs are shown in Table 2. Both the drug content and load-

ing efficiency of the RhB-loaded particles are greater than CF- loaded particles. These results are due to the interaction between the MSN particles and the dyes. The inside surface with APTES of MSN is negatively charged and RhB is posi- tively charged, which allows for easier binding of RhB mole- Table I − Particle size and zeta potential of MSN-based particles

(n=3).

MSN-based products Particle size (nm) Zeta potential (mV)

MSN 230.66±14.11 25.65±1.45

MSN-Tf 240.80±14.93 16.79±1.19

RhB/MSN 271.66±6.92 20.76±0.40

RhB/MSN-Tf 295.13±4.70 12.61±2.54

CF/MSN 276.03±4.19 19.31±0.73

CF/MSN-Tf 297.06±15.16 13.36±0.76

Fig. 2 − FE-TEM images of MSN (A) and MSN-Tf (B) at 30,000x magnification. Scale bars: 200 nm.

Table II − Drug content and loading efficiency of MSN-based particles (n=3).

MSN-based products Drug contents (% W/W)

Loading efficiency (% W/W)

RhB/MSN 6.54±0.80 92.04±5.09

RhB/MSN-Tf 6.04±0.37 90.53±3.17

CF/MSN 1.66±0.11 24.40±2.06

CF/MSN-Tf 1.20±0.12 18.28±2.62

cules to the inner surface of pores than CF.

This phenomenon causes CF to be easily washed and released from the MSN- based particles unlike with RhB. On the other hand, the drug contents and loading efficiencies of Tf-modified MSNs were a bit lower than that of unmodified MSNs because of the bulki- ness of Tf-modified MSNs.

Release properties of dye-loaded MSNs

Figure 3 shows the drug release behavior of RhB-loaded MSNs (Figure 3A), and CF-loaded MSNs (Figure 3B). To find out whether the release properties of dye-loaded MSNs could be affected by pH, the tests were conducted under two pH condi- tions: pH 7.4 (a, c, e and g) and pH 4.0 (b, d, f and h). An initial rapid release and a subsequent slow release profiles were observed in all systems. Of significance, neither the pH or Tf

modification affected the release properties of RhB-loaded MSNs. However, the release rates of RhB-loaded MSNs were considerably lower than MSNs loaded with CF, which may be due to the ionic interaction between RhB molecules and the inner surface of MSN, as mentioned before. Figure 3B shows that Tf modification may affect release rates to some degree, and this could be due to the surface-shielding effect that can cause a reduction in release rates. Additionally, the release rates are not affected by differences in pH.

Flow cytometry

The MSN uptake in HEK 293 cells and Jurkat cells was pro- ceeded by FACS. The cellular uptake of dye-loaded MSNs into difference cell lines are shown in Figure 4. In the graphs, the x-axis denotes the intensity of fluorescence and y-axis indi- cates cell count, while the gross area represents total cell count. Green (CF) and red (RhB) fluorescence were measured by FL1 (emissions wavelength 515-545 nm) and FL2 (564-606 nm), respectively. The cellular uptake of RhB-loaded MSNs and CF-loaded MSNs in HEK 293 cells are shown in Figures 4A and 4B. Similarly, the rates of cellular uptake of the same samples in Jurkat cells are shown in Figures 4C and 4D, respectively. Compared with control, the peaks of free RhB and CF shifted slightly in all cases. This suggests that there was some uptake of the free dyes in both cells. Further, in all Fig. 2 − Release properties of RhB- (A), CF- (B) loaded MSN-based

products. RhB/MSN and RhB/MSN-Tf were tested in pH 7.4 (a and c, respectively) and pH 4.0 (b and d, respectively).

CF/MSN and CF/MSN-Tf were tested in pH 7.4 (e and g, respectively) and pH 4.0 (f and h, respectively) in the same manner (n=3).

Fig. 4 − FACS analysis of dye-loaded MSN-based products. RhB and

CF was prepared in HEK 293 (A and B, respectively) and

Jurkat cells (C and D, respectively).

cases, the peaks of RhB- or CF-loaded MSNs shifted more than the free dyes. Therefore, the cellular uptake of the dyes was boosted by using MSN as a carrier. HEK 293 cells are not sensitive to transferrin, and correspondingly Tf modification had no apparent impact on cellular uptake in these cells, as seen in Figures 4A and 4B. On the other hand, in Jurkat cells in which transferrin receptors are overexpressed, the peaks of dye-loaded MSN-Tf did shift moderately compared to unmodi- fied MSN (Figures 4C and 4D). This indicates that the cellular uptake of Tf-modified MSNs increases considerably in TfR- expressing Jurkat cells compared to HEK 293 cells. Taken together, these data suggest that Tf modification provides unambiguous targeting capability to the dye-loaded MSNs.

Conclusion

There are several strategies that have been employed for targeted delivery of anticancer drugs. One is passive targeting like the EPR effect; and the other is active, which involves tar- geted ligands. MSN-Tf satisfies both strategies. We performed FT-NIR analysis to characterize and confirm the synthesis of MSN-Tf. Based on the Zeta-PSA results, our MSN-based parti- cles were suitable delivery systems that can allow for cellular uptake. Hydrophilic and hydrophobic drugs were loaded easily in the mesopores of MSN or MSN-Tf nanoparticles. Cellular uptake tests revealed that drugs loaded onto MSN-based parti- cles could be taken up more by the cells than the free drugs.

Specifically, the studies with transferrin-modified MSN reveal that transferrin could function as a targeting ligand. Collec- tively, these results suggest that our MSN-based particles would properly target specific cells and could be a suitable car- rier of anticancer drugs.

Acknowledgements

This study was financially supported by Chonnam National University.

References

1) Li, Y., Wang, J., Wientjes, M. G. and Au, J. L. : Delivery of nanomedicines to extracellular and intercellular compartment of solid tumor. Adv. Drug Deliv. Rev., 64, 29 (2012).

2) Xu, S., Olenyuk, B. Z., Okamoto, C. T. and Hamm-Alvarez, S.

F. : Targeting receptor-medicated endocytotic pathways with nanoparticles. Adv. Drug Deliv. Rev., 65,121 (2013).

3) Slowing, I., Vivery-Escoto, J. L., Wu, C. W. and Lin, V. S, : Mesoporous silica nanoparticles as controlled release drug delivery and gene transfection carriers. Adv. Drug Delivery Rev., 60, 1278 (2008).

4) Mekaru, H., Lu, J. and Tamanoi, F. : Development of mesoporous silica-based nanoparticles with controlled release capability fo cancer therapy. Adv. Drug Deliv. Rev., 95, 40 (2015).

5) Lu, J., Liong, M., Li, Z., Zink, J. I. and Tamanoi, F. : Biocompatibility, biodistribution, and drug-delivery efficiency of mesoporous silica nanoparticles for cancer therapy in animals. Small, 6, 1794 (2010).

6) Bharti, C., Nagaich, U., Pal, A. K. and Gulati, N. : Mesoporous silica nanoparticles in target drug delivery system. Int. J.

Pharm. Investig., 5, 124 (2015).

7) Moller, K. and Bein, T. : Talented mesoporous silcia nanoparticle. Chemistry of Materials, 29, 371 (2017).

8) Huang, I. P., Sun, S. P., Cheng, S. H., Lee, C. H., Wu, C. Y., Yang, C. S., Lo, L. W. and Lai, Y. K. : Enhanced chemotherapy of cancer using pH-sensitive mesoporous silica nanoparticles to antagonize P-glycoprotein–mediated drug resistance.Mol.

Cancer Ther., 10, 761 (2011).

9) Brannon-Peppas, L. and Blanchette, J. O. : Nanoparticle and targeted systems for cancer therapy. Adv. Drug Delivery Rev., 64, 206 (2012).

10) Lee, C. H., Cheng, S. H., Huang, I. P., Souris, J. S. Yang, C. S., Mou, C. Y. and Lo, L. W. : Intracellular pH-responsive mesoporous silica nanoparticles for the controlled release of anticancer chemotherapeutics. Angew. Chem., Int. Ed., 49, 8214 (2010).

11) Daniels, T. R., Bernabeu, E., Rodriguez, J. A., Patel, S., Kozman, M., Chiappetta, D. A., Holler, E., Ljubimova, J. Y., Helguera, G. and Penichet, M. L. : The transferrin receptor and the targeted delivery of therapeutic agents against cancer.

Biochim. Biophys. Acta, 1820, 291 (2012).

12) Kovár, J. 1, Seligman, P. and Gelfand, E. W. : Lymphocyte lines under iron-depriving conditions: transferrin receptor expression related to various growth responses. Immunol. Lett., 42, 123, (1994).

13) Wang, J., Tian, S., Petros, R. A., Napier, M. E. and Desimone, J. M. : The complex role of multivalency in nanoparticles targeting the transferrin receptor for cancer therapies.J. Am Chem. Soc., 132, 11306 (2010).

14) Suteewong, T., Sai, H., Bradbury, M., Estroff, L. A., Gruner, S.

M. and Wiesner, U. : Synthesis and formation mechanism of

aminated mesoporous silica nanoparticles. Chem. Mater.,24,

3895 (2012).

15) Wang, Y., Shi, W., Song, W., Wang, L., Liu, X., Chen, J. and Huang, R. : Tumor cell targeted delivery by specific peptide- modified mesoporous silica nanoparticles. J. Mater. Chem., 22, 14608 (2012).

16) Lee K. M, Kim I. S, Lee Y. B, Shin S. C, Lee K. C. and Oh I.

J. : Evaluation of transferrin-polyethylenimine conjugate for targeted gene delivery. Arch. Pharm. Res., 28, 722 (2005).

17) Song, B., Wu, C. and Chang, J. : Dual drug release from electrospun poly(lactic-co-glycolic acid)/mesoporous silica nanoparticles composite mats with distinct release profiles. J.

Control Release, 145, 257 (2010).

18) Bae, Y. H. and Park, K. : Targeted drug delivery to tumors:

Myths, reality and possibility. J. Control. Release, 153, 198 ( 2011).

19) He, C., Hu, Y., Yin, L., Tang, C. and Yin, C. : Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials, 31, 3657 (2010).

20) Lu, J., Liong, M., Zink, J. I. and Tamanoi, F. : Mesoporous silica nanoparticles as a delivery system for hydrophobic anticancer drugs. Small, 3, 1341 (2007).

21) Liu, Y., Miyoshi, H. and Nakamura, M. : Novel drug delivery

system of hollow mesoporous silica nanocapsules with thin

shells: preparation and fluorescein isothiocyanate (FITC)

release kinetics. Colloids Surf B Biointerfaces, 58, 180 (2007).