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Silver coated colloidosomes as carriers for an anticancer drug Qian Sun, Hui Gao, Gleb B. Sukhorukov, and Alexander Francis Routh ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b11128 • Publication Date (Web): 07 Sep 2017 Downloaded from http://pubs.acs.org on September 8, 2017

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ACS Applied Materials & Interfaces

Silver coated colloidosomes as carriers for an anticancer drug Qian Sun 1*, Hui Gao 2*, Gleb B. Sukhorukov 2, Alexander F. Routh 1†

AUTHOR ADDRESS 1. Department of Chemical Engineering and Biotechnology, University of Cambridge, West Cambridge Site, Philippa Fawcett Drive, CB3 0AS, Cambridge, United Kingdom. 2. School of Engineering and Materials Science, Queen Mary University of London, Mile End Road, E1 4NS, London, United Kingdom.

KEYWORDS: Colloidosomes, Silver shells, Impermeable, Ultrasound, Drug carries.

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ABSTRACT

Small drug molecules are widely developed and used in the pharmaceutical industry. In the past few years, loading and delivering such molecules using polymer shell colloidosomes has attracted interest. Traditional polymer capsules fail to encapsulate low molecular weight materials for long times, since they are inherently porous and permeable for small molecules. In this paper, we report a method for encapsulating an anticancer drug with small molecule weight, for cell viability tests. The silver coated colloidosomes are prepared by making an aqueous core capsule with a polymer shell and then adding AgNO3, surfactant and L-ascorbic acid to form a second shell. The capsules are impermeable and can be triggered using ultrasound. We propose to use the capsules as drug carriers. The silver demonstrates a low cytotoxicity for up to ten capsules/cell. After triggering the silver shells by ultrasound, the released doxorubicin, the broken silver fragments, and the doxorubicin loading on the capsule surface all kill cells. The results demonstrate a non-permeable silver shell microcapsule with ultrasound sensitivity for potential medical applications.


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INTRODUCTION

Small drug molecules play an important role in medical applications and are widely used in the pharmaceutical industry 1, 2. In order to deliver such drugs to targeted areas, microcapsules have been developed and used

3-7

. Water core colloidosomes are

capsules, which are able to seal and deliver low molecular weight encapsulated materials and release them at desired points. They have become a rapidly growing research area 8-12. Various methods have been reported to prepare water core microcapsules, such as using self-assembly of colloidal particles

13, 14

, a sacrificial template

templated layer-by-layer polyelectrolyte self-assembly

17, 18

15, 16

, and

. The technique of self-

assembly of colloidal particles at the interface between oil and water phases is conceptually simple. It involves emulsification of an aqueous dispersion of colloidal polymer particles in an organic phase, with the applied shear rate governing the size of the emulsion droplets formed. The colloidal particles migrate to the oil/water interface and stabilize the emulsion droplets. The formed capsules then require stabilization by locking the particle layer

19-21

. The first colloidosomes were made by Velev et al.

and further developed by Dinsmore et al. 25, Cayre et al. et al.

28, 29

, Routh et al.

30, 31

22-24

26

, Biggs et al. 27, Sukhorukov

and other researchers 32, 33. An advantage of these capsules

is that they can be formed in mild conditions. In addition, after modification, they can be magnetic 34, temperature 35 and pH-responsive 36. However, the encapsulation of small molecules within polymer shells still presents a significant challenge. Most polymer capsule shells are leaky to small molecules, due to their intrinsic high permeability. This greatly limits their practical application

37, 38

. In a previous study, we synthesized impermeable silver coated

colloidosomes by leaching L-Ascorbic acid from the core and washing with a silver ACS Paragon Plus Environment

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nitrate (AgNO3) solution

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31

. The use of silver makes the capsules impermeable and

enables the use in biological applications. In this paper, we report an improvement on the silver shell making process, leading to a higher loading efficiency and the successful loading of an anticancer drug. We also report cell viability results, and the release of the therapeutic by ultrasound. We believe there are medical applications for silver coated colloidosomes, as carriers for small hydrophilic drugs.

EXPERIMENTAL SECTION

Materials. Colloidal polymer particles of diameter 153 nm, as determined by dynamic light scattering (Brookhaven ZetaPALS), were made by conventional emulsion polymerisation. The monomers used were methyl methacrylate and butyl acrylate30. The relative amounts of the different monomers controlled the glass transition temperature which was found, using differential scanning calorimetry, to be 35oC. The chemicals used in the experiments were sodium dodecyl sulfate (SDS, Fisher Scientific), Allura Red AC dye (Sigma-Aldrich), Buffer solution pH 10.0 (Sigma-Aldrich),

4,4’-dithiodibutyric

acid

(DDA,

95%,

Sigma-Aldrich),

and

Doxorubicin hydrochloride anticancer drug (98.0%, Sigma-Aldrich). They were all used as received without purification. For cell culture and viability studies the materials used were Dulbecco’s modified eagles media (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, trypsin, thiazolyl blue tetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO). These were all supplied by Sigma-Aldrich. Ultrapure deionised water, of resistivity 18.2 MΩ•cm, was produced by a Pure Lab Ultra apparatus. For mixing, a TopMix FB15024 vortex mixer (Fisher Scientific) was used. To purify the synthesised colloidosomes, dialysis tubing with a MWCO of 1200014000 (Spectrum Laboratories) was employed. ACS Paragon Plus Environment

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To make polymer shell colloidosomes, a method from a previous paper was followed31. A Silverson high shear mixer (model SL2) was used to mix 200 mL of sunflower oil, 4 mL of Span 80 and 2 mL of a 5.6 wt% latex particle suspension. After emulsification the mixture was heated at 50 °C for 1 h. The mixture was then centrifuged and redispersed in water. The formed capsules had a diameter between 0.7 µm and 2 µm, with an average of 1.2 µm and the shell appeared smooth. Doxorubicin encapsulation. Figure 1 shows schematically how silver coated colloidosomes loading doxorubicin hydrochloride were formed. A Silverson high shear mixer (model SL2) was used to mix 200 mL sunflower oil and 4 mL of Span 80 in a 400 mL beaker. A separate aqueous phase containing 5.6 wt% latex particles and 2 mg/mL doxorubicin hydrochloride in a pH 10 buffer solution was made and 2 mL of this aqueous phase was added into the sunflower oil, whilst applying shear. Five cycles of mixing and rest were applied, where shear was applied for 60 s, followed by 30 s rest. To anneal the polymer shells the emulsion mixture was heated in a water bath. To ensure rapid heat transfer the emulsion mixture was split into five different tubes and when the temperature reached 50 ± 0.5°C, the emulsions were heated for 1 h. To clean the capsules, 20 ml of the solution was centrifuged at 20°C and 3000 rpm for 10 min. After removing the vegetable oil, 24 mL of a 0.1 wt % aqueous solution of AgNO3 and 2 mL of a 1 wt% aqueous solution of SDS were added and the mixture was agitated using the vortex mixer. Addition of 2 mL of a 15 wt% L-ascorbic acid aqueous solution started the silver reduction reaction, which was allowed to proceed for 1 h. To obtain the final aqueous dispersion of silver coated microcapsules, the mixture was centrifuged, at 20°C, at 1500 rpm for 2 min followed by redispersal in a 0.1 wt% SDS solution.


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To allow biotargeting of the capsules, a surface modification with 4,4’dithiodibutyric acid (DDA) was carried out. A 20 mL solution of 0.5 wt% 4,4’dithiodibutyric acid (DDA) in ethanol was mixed using the vortex and a known mass of silver coated colloidosomes was added. Mixing by a magnetic stirrer for 48 h at room temperature allowed the reaction to proceed, after which the modified capsules were cleaned by five cycles of centrifuging at 1500 rpm for 2 min followed by redispersal in ultrapure water. Dye encapsulation and dye release. The steps followed to encapsulate the dye solution were the same as above. The organic phase was 200 mL of sunflower oil and 4 mL of Span 80. The aqueous phase was 2 mL of a dispersion of 5.6 wt% latex particles and 2 wt% Allura Red. To measure the dye release, 20 ml samples were put into dialysis tubing which were then put into a larger bottle containing 450 mL of water. A magnetic stirrer was employed and 0.5 mL samples were taken for spectroscopic characterization at regular intervals. Release by ultrasonic treatment. An ultrasonic probe, operating at a frequency of 20 kHz and power output of 50 W was used. Different times of application were employed using an ultrasonic processor GEX 750 (Sonics & Materials Inc., USA). The temperature change was kept to below 5°C by using an ice bath. Cell Culture and Cell Viability Test. DMEM with 10% FBS and penicillinstreptomycin (1%) was used in an atmosphere of 5% CO2/95% air at 37°C to culture rat neuroblastoma B50 cells. A standard MTT assay was used to assess cell viability, using a BMG Fluostar Galaxy plate reader. The cells were initially plated at 20 000 cells per well on 96 well plates. After a day, capsules at ratios of 10, 20, or 50 capsules per cell were added to triplicate wells. Incubation times of 1, 2, or 3 days were used and after this time each well had 100 µL of thiazolyl blue tetrazolium bromide (MTT) solution (5 mg/mL in cell culture medium) added. The plates were shaken and then ACS Paragon Plus Environment

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incubated for 3 h before addition of dimethyl sulfoxide (DMSO). The cell number was then read by a Multiskan Ascent plate reader. Sample characterizations. A Zeiss X-beam FIB SEM was used to image the colloidosomes, using a voltage of 5.0 kV. The silver coated colloidosome samples were imaged without any treatment after air-drying a drop a stainless steel SEM stub. The same instrument was used for energy-dispersive X-ray spectroscopy (EDX), to determine the elemental composition of colloidosomes. In this case an accelerating voltage of 10.0 kV was used. Transmission electron microscopy (TEM) was performed using a FEI Philips Tecnai 20 TEM at 200 keV (Tungsten, LB6). Fluorescent images were captured with a Nikon EFD-3 fluorescence microscope by a flourescein isothiocyanate filter. A Leica TS confocal scanning system (Leica, Germany) equipped with a 63×/1.4 oil immersion objective was used to obtain confocal laser scanning microscopy (CLSM) images. Spectrometry was carried out using a UV-vis spectrophotometer equipped with a tungsten lamp (Thermo Scientific, model Heλios Gamma). Samples were placed in polystyrene cuvettes of 10 mm path length. Ultrapure water was the reference material and the absorbance value at 500 nm was recorded.

RESULTS AND DISCUSSION

Dye encapsulation. Initially, we used silver coated colloidosomes loading Allura Red AC dye solution as a model. The reason why we used Allura Red AC is that this dye does not affect the colloidosomes making process, it is small enough to diffuse from the porous polymer shells and it is not too pH sensitive. Figure 2 shows SEM, TEM, EDX and fluorescent pictures of the colloidosomes with silver shells, which contained dye. As can be seen, the polymer shells are fully covered by silver particles. ACS Paragon Plus Environment

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The capsule diameter is between 0.9 µm and 3.2 µm. Figure 2d shows the EDX image of the capsules. The large silver peaks suggest silver particles are surrounding the colloidosome surface. The strong carbon peak arises from the polymer shell. The SEM stub is responsible for the aluminum peak and the other weak peaks arise from the surfactant. It can be seen from the fluorescent image in Figure 2e that the dye solution can be successfully sealed in the colloidosomes. Figure 3 shows the release profiles of Allura Red dye from silver shell, polymer shell or unsealed colloidosomes. The release data was normalized by the amount of encapsulated dye. This is the amount of dye added to the experiment minus the amount measured in the waste oil and wash solutions. The calculation of the encapsulation efficiency and dye loss data are shown in supporting information table S1. Figure 3 illustrates the dye release profiles for the blank tests. It can be observed that all three control samples (sample 1: dye solution, sample 2: dye solution and latex particles, and sample 3: dye solution, latex particles and silver particles) released dye without application of any trigger. It is noticeable that the three control samples display similar release trends. . The release rate from polymer shell colloidosomes (sample 4) was slower than the three control samples. In addition less material was finally released which indicates that some dye was absorbed onto the latex particles, or possibly that some dye was lost during the manufacturing process. In our pervious study, we demonstrated that the dye release of the silver shell was negligible and silver shell can be triggered adding nitric acid. Previously we reported an encapsulation efficiency of 20%

31

. Briefly, the previous colloidosomes were

prepared by leaching L-Ascorbic acid from the core to react with silver nitrate (AgNO3) in the wash solution, forming silver on the surface on the polymer shells. In order to improve the loading efficiency, the new silver shells can be prepared by making the polymer shell capsules first, then redispersing them in AgNO3 solution, and ACS Paragon Plus Environment

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adding L-Ascorbic acid. It can be seen that there was very little dye on the outside of the new method colloidosomes (sample 5). We obtain a higher loading efficiently using the new method. When 5 mL 1 wt% nitric acid was added, dissolving the shell (sample 6), the measured release yield was 47 %. It should be noted that the high temperature (65 °C) and nitric acid destroys some of the dye lowering the measured efficiency. When ultrasound treatment was used to break the silver shell colloidosomes (sample 7), a release yield of around 8.5% was measured. The reason for such a low value is that when the colloidosomes are too crowded, only the capsules near the ultrasonicator probe can be triggered. The release results demonstrate complete sealing of the capsules and that dye release can be achieved using nitric acid or ultrasound. Release by ultrasonic treatment. A suspension of microcapsules was subjected to ultrasound sonication at 20 kHz for different durations. Figure 4 shows the SEM images of silver shell colloidosomes after various sonication times. Some of the capsules were seen to be broken into fragments after 120 s of ultrasound treatment and most of the capsules were destroyed after 240 s. For longer sonication times, more capsules were broken. After 480 s of sonication, only a few colloidosomes survived and there were a large number of small pieces of broken shell. For the ultrasound treatment with a high power of 50 W, a few minutes is enough to break most of the silver coated colloidosomes and release the dye solution. However, considering of the application, we also tried high intensity focused ultrasound (HIFU) with a frequency of 2.65 MHz and a power of 6 W, which can be used for cancer treatments. Figure 5 shows the SEM images of silver shell colloidosomes after 120 s of HIFU treatment. It is clearly seen that a part of the silver shells were deformed and broken after exposing of HIFU for 120 s, however, there were still a large number of intact colloidosomes. This may be caused by the lower


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power of the ultrasound, where a relatively long duration time is needed to damage the silver coated colloidosomes. Drug encapsulation. Figure 6 shows SEM images of colloidosomes with a silver shell loading doxorubicin hydrochloride and the same capsules after 120 s of ultrasound treatment. Figure 7 shows CLSM images of the same capsules before and after ultrasound treatment. Before ultrasound treatment the silver particles fully cover the polymer shells and seal the fluorescent doxorubicin in the core. After 120 s of ultrasound treatment, most of the capsules were destroyed. There were a large number of small dots appearing in the CLSM images, which are likely to be the small pieces of broken shell and released doxorubicin. Figure 8 shows CLSM images of the surface of some capsules loading doxorubicin. When focusing onto the surface of the capsules, there was also some fluorescent doxorubicin appearing, which cannot be easily washed off. This indicates that the drug was not only sealed in the silver shells, but some was also attached on the surface of the capsules during the manufacture. Table 1 shows the calculation of the drug encapsulation efficiency. The original total doxorubicin added in the experiment was 100%. During the capsule making process, 27.75% of the total drug was lost in the sunflower oil and 49.75 % of the total drug was lost in the washing solution. So the remaining drug loading in the capsules was about 22.5%. Cell viability test. A cytotoxicity study was carried out in vitro at different capsules/cell concentrations for B50 cancer cells for up to 72 h. Figure 9 shows the viability of the B50 cells when mixed with different amounts of polymer shell colloidosomes, free doxorubicin, silver shell colloidosomes, silver shell colloidosomes after 120 s ultrasound, silver shell colloidosomes loading doxorubicin and silver shell colloidosomes loading doxorubicin after 120 s ultrasound. The experiments were carried out for (a) 24 h, (b) 48 h, and (c) 72 h at 37 °C, and measured by an MTT assay ACS Paragon Plus Environment

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relative to the control. For broken silver shells, the ultrasound was used for 120 s at a frequency of 20 kHz and power output of 50 W, before adding into the B50 cells. As seen in Figure 9, for 10 capsules/cell, both the polymer shells and free doxorubicin show low toxicity to the cancer cells at a cell viability more than 95% from 24 h to 72 h. Silver shells results indicate low cytotoxicity, in Figure 9a, showing the cell viability at more than 90% during the first 24 h and in Figure 9b more than 80% after 48 h. After 72 h, in Figure 9c, the cell viability of silver shells dropped to around 60%, indicating an increased toxicity to cells when prolonging the incubation time. After ultrasound treatment, the broken silver shells display a relatively high cytotoxicity with a cell viability of around 50% during the first 48 h and around 40% after 72 h. The possible reason is that after ultrasound the pieces of broken shell are smaller compared with intact capsules, which become more toxic to living cells 39-42. The silver shell colloidosomes loading doxorubicin also lead to a low cell viability, showing around 20% after 72 h in Figure 9c. A possible reason for this is that when making the silver shell colloidosomes, the drug may diffuse through the polymer shell and grow together with the silver shell. This may cause some doxorubicin to be attached to the surface of the silver shell and it is not easily washed off. The doxorubicin on the surface may then be toxic to the cancer cells, leading to the decreased cell viability. The silver shells loading doxorubicin after 120 s of ultrasound treatment illustrate a higher cytotoxicity, displaying a viability of about 15% after 72 h. This is because of the released drug, the broken silver shells and the drug attached on the surface of the capsules all killing the cancer cells. The cell viability tests of 20 capsules/cell, display a similar trend. Both the polymer shells and free doxorubicin show low toxicity to cancer cells. However, the silver shell results indicate a higher cytotoxicity compared with polymer shells, in Figure 9b, showing the cell viability at about 60% during the first 48 h. After 72 h, in ACS Paragon Plus Environment

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Figure 9c, the cell viability of silver shells dropped to around 50%. After ultrasound treatment, the broken silver shells display a relatively high cytotoxicity, showing a cell viability of around 40% during the first 48 h and lower than 30% after 72 h. The silver shell colloidosomes loading doxorubicin also lead to a low cell viability, showing around 20% after 72 h in Figure 9c. After 120 s of ultrasound treatment, it illustrates a higher cytotoxicity, displaying a viability of about 5% after 72 h. The cell toxicity of silver shell capsules was significantly higher, than the polymer shell capsules, when the concentration reached 50 capsules/cell. The free doxorubicin at this concentration is more toxic than 10 and 20 capsules/cell, showing a cell viability of 85% after 24 h, 78% after 48 h and 86% after 72 h. An increase in the amount of silver and doxorubicin means the cytotoxicity is increased. The silver shell capsules display a viability value of about 20% after 72 h in Figure 9c. After ultrasound treatment, the broken silver shells display a cell viability of around 15% after 72 h in Figure 9c. Both the silver shell colloidosomes loading doxorubicin and the ones after ultrasound lead to a low cell viability, showing around 5% after 72 h. According to the cell viability results, all the capsule concentration, size and composition can determine the final proliferation and death rate of cells. After triggering the silver shells using ultrasound, the combined effects of the released doxorubicin, the broken silver fragments and the drug attached on the surface of the capsules can kill cancer cells. It is also clear that the amount of the capsules is also a crucial design parameter.

CONCLUSIONS

Our study has demonstrated a method for encapsulating cargos using silver coated colloidosomes. The colloidosomes are impermeable for small molecules and can be ACS Paragon Plus Environment

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triggered using ultrasound. After 120 s of ultrasound treatment, most of the capsules were destroyed into small pieces and only a few survived. We propose the possibility to use the capsules for delivery of anticancer drugs, i.e., doxorubicin. For 10 capsules/cell, the silver shells have a low cytotoxicity, showing a cell viability of more than 60% after 72 h. After triggering the silver shells, the triggered doxorubicin, broken silver fragments and the drug attached on the capsule surfaces kill cells. The number and the size of the capsules is crucial for their efficacy.

AUTHOR INFORMATION

Corresponding Author † Dr. Alexander F. Routh E-mail address: [email protected], Tel: 01223765718, Fax: 01223765701 Department of Chemical Engineering and Biotechnology, University of Cambridge, West Cambridge Site, Philippa Fawcett Drive, CB3 0AS, Cambridge, United Kingdom Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. * These authors contributed equally.

ASSOCIATED CONTENT

SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS Publications website. The calculation of the encapsulation efficiency and dye loss data are shown in the supporting information.


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ACKNOWLEDGMENT

The authors thank Dr Richard Langford and Eric Tapley (The Cavendish Laboratory, Department of Physics, University of Cambridge) for their assistance with use of the scanning electron microscopy and the transmission electron microscopy. Many thanks for Ziyan Zhao and Lisa Hall (Department of Chemical Engineering and Biotechnology, University of Cambridge) for their assistance with use of the fluorescent microscope. Dong Luo is thanked for assistance with the free doxorubicin cell viability tests. Qian Sun and Hui Gao are grateful to the China Scholarship Council for funding.


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FIGURE CAPTION:

Figure 1. Fabrication of colloidosome with a silver shell loading doxorubicin hydrochloride anticancer drug. Figure 2. (a) (b) SEM, (c) TEM, (d) EDX and (e) fluorescent microscope images of the silver coated colloidosomes loading dye solution. Figure 3. Profiles shwing the release of Allura Red dye from colloidosomes with and without a a silver shell. Error bars of the standard error of measurements, typically ±∼3%, are not shown for clarity. Figure 4. SEM images of silver shell colloidosomes after various sonication times: (a) untreated; (b) 60 s; (c) 120 s; (d) 240 s and (e) 480 s. Figure 5. SEM images of colloidosomes with a silver shell after 120 s HIFU; (a) – (f) showing different magnifications. Figure 6. SEM images of colloidosomes with a silver shell loading doxorubicin hydrochloride anticancer drug and the same capsules after 120 s ultrasound treatment; (a) – (f) showing different magnifications. Figure 7. CLSM images of silver coated colloidosomes loading doxorubicin hydrochloride with λex =480 nm, doxorubicin (left), bright field (middle), and merged (right) images. (a) Before ultrasound and (b) After 120 s of ultrasound treatment. Figure 8. CLSM images of the surface of capsules loading doxorubicin. Figure 9. Viability of B50 cells mixed with polymer shell colloidosomes at different concentrations of, free doxorubicin, silver shell colloidosomes, silver shell colloidosomes after 120 s ultrasound, silver shell colloidosomes loading doxorubicin and silver shell


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colloidosomes loading doxorubicin after 120 s ultrasound capsules for (A) 24 h, (B) 48 h, and (C) 72 h at 37 °C. The error bars show the standard deviations.


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Figure 1:

Figure 2:


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Figure 3:

Figure 4:


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Figure 5:

Figure 6:


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Figure 7:

Figure 8:


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Figure 9:

Table 1. The calculation of the drug encapsulation efficiency Drug data

Mass

Percentage

Amount of drug added in the experiment

4.0 mg

100 %

Amount measured in the sunflower oil

1.11 mg

27.75 %

Amount measured in the washing solution

1.99 mg

49.75 %

Amount remaining in or on the capsules

0.90 mg

22.50 %


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REFERENCES

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