Doxorubicin carriers based on Au nanoparticles – effect of shape and gold-drug linker on the carrier toxicity and therapeutic performance

Abstract

Gold nanoparticles (AuNPs) prepared by the Turkevich method and near-IR absorbing non-spherical anisotropic nanotriangles (AuNTs) prepared by the thiosulfate method were used for doxorubicin binding. The drug was connected to the polyethyleneglycol modified gold nanoparticle by covalent peptide or pH-active hydrazone linkers. Their optical properties were studied by UV-vis spectroscopy. The shape and size of the nanoparticles were evaluated by SEM and DLS. Fluorescence studies demonstrated different drug release profiles depending on the pH. An MTT assay performed on two cell lines (A549 and HeLa) revealed that gold nanostructures modified with doxorubicin were more toxic than free doxorubicin. Viability tests and confocal microscopy revealed that the bond between the Au carrier and the drug determined the pathway of cell death – apoptotic in the case of peptide bonding and sudden and necrotic when a hydrazone linker was employed.

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Introduction

Gold nanoparticles (AuNPs) have attracted increasing interest in chemistry, biomedicine and electronics due to their controllable small size, oxide-free surface, unusual optical and electronic properties, easy bioconjugation, stability and good biocompatibility.^1,2 Appropriate size management provides control over several chemical and physical properties, e.g. small gold nanoparticles (AuNPs) mainly absorb light, while for those above 80 nm, the ratio of scattering to absorption increases. As the size increases higher-order modes at lower energies become dominant. This causes a red-shift and broadening of the surface plasmon band.³ AuNPs are being explored to induce hyperthermic cytotoxicity.⁴ Once exposed to light of a suitable wavelength, the conduction-band electrons of NPs generate heat that is transmitted to the cells and surrounding tissues. Various energy sources can be applied, including radio frequencies, high intensity focused ultrasound, microwaves and lasers. This thermal therapy has the advantage of killing cancer cells without causing resistance and regardless of the genetic background enabling it to be applied to all cancer patients.⁵ Recently, non-spherical anisotropic nanotriangles (AuNTs), which not only exhibit the properties of solid AuNPs, but also the unique optical properties in the near-infrared region (NIR), were described.⁶ Gold/gold sulfide (GGS-NPs) composite core–shell structures showing strong NIR absorption were first produced by Zhou et al.⁷ through a two-step reduction of HAuCl₄ by Na₂S. The optical properties of these materials were later explained to be due to a metal-coated dielectric nanoparticle structure.⁸ In 2007, Schwartzberg et al. observed that Na₂S solution is not stable during the ageing process,⁹ but can convert to different sulfur-containing compounds. One of them is sodium thiosulfate – the reagent for producing GGS-NPs. The author suggested that the structure of the main product in the reaction of chloroauric acid with sodium sulfide is not a core–shell structure, but rather triangular, icosahedral and rod-like gold aggregates. Later, in 2012, Zhang et al. synthesized gold anisotropic nanotriangles (AuNTs) by a single-step reaction of HAuCl₄ with Na₂S₂O₃ without any additional templates, capping agents or seeds.⁶ Due to the low absorption and high transmission of NIR light by the tissue chromophores, less damage is caused to human cells compared with shorter wavelengths of light. This makes the NIR-absorbing non-spherical AuNPs suitable for biomedical applications, such as imaging, detection and thermal therapy of cancer.^10–15 In the present study, two approaches were used to synthesize gold nanoparticles as supports for the anticancer drug – doxorubicin: the thiosulfate method reported by Zhang et al.⁶ to prepare NIR-sensitive nanotriangles (AuNTs) and the Turkevich¹⁶ method to obtain spherical gold nanoparticles (AuNPs). Doxorubicin, also called daunomycin or Adriamycin is an anthracycline antibiotic. The first anthracyclines were isolated from the pigment-producing Streptomyces peucetius in the early 1960s. Currently, they are commonly used in the treatment of a wide range of cancers.¹⁷ New carriers for drug targeting have been developed to improve the selectivity and efficiency of drug release.^18,19

Studies of tumour cells have shown that the pH of pathologically changed cells is lower than that of healthy ones. This difference has been taken into account in designing pH-dependent drug carriers. Our aim was to compare gold nanoparticles modified with doxorubicin using either a bifunctional PEG cross-linker to create an amide bond (Scheme 1A) or a PEG cross-linker and hydrazine to form a pH-sensitive hydrazone bond (Scheme 1B).

Scheme 1 (A) Model of gold nanoparticles modified with doxorubicin by a peptide bond. (B) Model of gold nanoparticles modified with doxorubicin by a hydrazone bond.

The hydrazone bond has attracted some interest of the researchers.^20,21 Despite its stability in neutral and alkaline pH, in acidic media, the bond is destroyed with the formation of free hydrazide.²² Therefore, it is possible to use the hydrazone bond for the construction of anthracycline drug carriers for the pH-triggered release of the drug.²³ Aryal et al. reported DOX-conjugated thiol-stabilized Au nanoparticles with hydrazone bonds.²⁴ Etrych et al. synthesized copolymer structures as drug carriers with DOX attached to the polymer via pH-sensitive or amide linkers.²⁵

In our approach, PEG moieties allow coupling with the target drug, but also prevent the nanoparticles from aggregation. Among the studied gold nanostructures (AuNSs) (Scheme 1): AuNTs-PEG-DOX, AuNPs-PEG-DOX and AuNPs-PEG-Hyd-DOX, NIR-sensitive AuNTs modified with DOX are very interesting because they could be considered as bifunctional therapy agents – not only thermal generating agents but also efficient drug carriers. In the case of AuNPs-PEG-Hyd-DOX and AuNPs-PEG-DOX, the core parts are the same and only the linkers are different – either pH-sensitive or insensitive. The interactions of drug-modified NPs with biological material were examined by confocal microscopy and the MTT assay and led to conclusions concerning the role of the hydrazone bond present in the linker part.

Experimental

Materials and methods

Chemicals and reagents. Doxorubicin hydrochloride salt was purchased from LC Laboratories (Woburn, Massachusetts, USA). Other compounds used in this work for the syntheses were purchased from Aldrich (Poland) and Fluka (Poland). Britton–Robinson buffers were prepared using water from a Hydrolab ultrapure water system via the addition of appropriate amounts of 0.2 M sodium hydroxide to acetic acid, boric acid and orthophosphoric acid (0.04 M solutions). The pH was measured using a pH-Meter E2 (Mettler Toledo).

Spectroscopic measurements. UV-vis spectroscopic measurements were performed using a UV-vis EVOLUTION60 spectrophotometer (Thermo Scientific, Waltham, Massachusetts, USA) with a 1 cm quartz cell in the wavelength range from 400 to 1100 nm.

Fluorescence spectroscopy measurements. Fluorescence spectroscopy measurements were performed using the SPEX Fluorolog 3 spectrometer (Horiba Scientific Ltd., Edison, New Jersey, USA). Drug release studies were performed using 470 nm excitation (3 nm bandpass). The amount of free doxorubicin was monitored by recording emissions between 500 and 700 nm (3 nm bandpass) with a DOX maximum at 590 nm. The kinetics of the doxorubicin release were measured for AuNPs-PEG-Hyd-DOX solutions with a 5.8 × 10⁻² μM concentration of DOX in a 1 cm length acrylic cell. For these measurements, the pH of the solutions was 7.4 and 5.5.

Scanning electron microscopy measurements. Scanning electron microscopy measurements were performed on a MERLIN Field Emission Scanning Electron Microscope with a Gemini II column using a BSE detector (Carl Zeiss, Cambridge, UK).

Dynamic light scattering measurements. Dynamic light scattering measurements were performed on a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK) in a 1 cm quartz cuvette. The specimens were diluted with ultrapure distilled water. The angle of the incident light was 173°. Each specimen underwent 5 measurements, and the mean size was calculated.

Confocal microscopy. Confocal microscopy was used to determine the type of cell death caused by the NPs-DOX treatment. A549 cells (3 × 10⁵ per well) were seeded on 8-well Lab-Tec II™ live cell imaging chambered cover glasses (Thermo Scientific, Waltham, Massachusetts, USA) a day before the addition of the different AuNPs carriers bearing immobilized DOX to achieve the calculated final DOX concentration of 3 μM. After the addition of nanomaterials, pictures were taken every 15 min with a NIKON A1 confocal microscope equipped with NIS ELEMENTS software. A NIKON ×60 Plan Fluor objective was used. Fifteen minutes before imaging, the nuclei were stained with Hoechst 33342 dye at a concentration of 2 μg mL⁻¹. The DOX fluorescence was excited with 561 nm laser light and observed with a 570–620 nm emission filter.

Preparation and characterization of DOX-tethered AuNPs (AuNPs-PEG-DOX)

Briefly, spherical gold nanoparticles (AuNPs) were prepared by the Turkevich method by the citrate reduction of chloroauric acid.¹⁶ In this method, 0.1 mmol of chloroauric acid was added to 100 mL of double-distilled water and brought to a boil. Next, 10 mL of citric acid (0.22 mmol in H₂O) was quickly added to the solution. The refluxing of the solution continued until the colour of the boiling solution changed from dark purple to a red wine colour. The mixture was boiled with stirring for 10 more minutes and allowed to cool in the dark. The nanoparticles were roughly spherical, and the average diameter was approximately 50 nm, as determined by UV-vis spectroscopy and DLS analysis (Fig. 1 and Table 1). The as-prepared nanoparticles were modified with doxorubicin as follows: 2 μmol of O-(2-carboxyethyl)-O′-2-mercaptoethylhepta-ethylene glycol (HOOC-PEG-SH) was dissolved in 1 mL of water and added to 100 mL of freshly prepared citrate AuNPs. The solution was stirred for 2 hours at room temperature in the dark.^26,27 Next, doxorubicin hydrochloride, EDAC (2 μmol each) and 1 μL of triethylamine were dissolved in 1 mL of water and added to the stirring solution. After 2 h, the precipitate of AuNPs-PEG-DOX was centrifuged, separated out, washed several times with distilled water and dissolved in a small amount of DMSO, Scheme 2A.

Fig. 1 UV-vis spectra of gold nanoparticles protected by: (1) sodium citrate (AuNPs), (2) O-(2-carboxyethyl)-O′-(2-mercapto-ethyl)heptaethylene glycol before (AuNPs-PEG) and (3) after binding DOX (AuNPs-PEG-DOX).

Table 1 Sizes of AuNPs-PEG, AuNTs-PEG, AuNPs-PEG-DOX and AuNTs-PEG-DOX measured by dynamic light scattering (DLS)

	AuNPs-PEG	AuNPs-PEG-DOX	AuNTs-PEG	AuNTs-PEG-DOX
Size [nm]	53.9 ± 3.3	58.9 ± 1.6	70.7 ± 5.2	81.8 ± 4.7

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Scheme 2 Pathways of syntheses of AuNPs-PEG-DOX (A), AuNPs-PEG-Hyd-DOX (B) and AuNTs-PEG-DOX (C).

The concentration was determined by weighing the solid content after the solvent evaporation and was found to be 5.8 mg mL⁻¹. The solution was stable even after a hundred-fold dilution with water and acidic/basic pH buffers. No aggregation during synthesis or after modification with thiolated PEG and DOX was observed, as indicated by no change in the maximum absorption in the UV-vis spectra (Fig. 1), and the size before and after modification was controlled by dynamic light scattering spectroscopy and scanning electron microscopy, (Fig. 2A and B and Table 1).

Fig. 2 FE-SEM images of AuNPs-PEG-DOX (A and B) and AuNTs-PEG-DOX (C–E). Images of AuNTs-PEG-DOX before (C) and after (D) separation. Size distributions of AuNPs-PEG-DOX (B) and AuNTs-PEG-DOX (E).

The concentration of doxorubicin on the AuNPs surface was determined by UV-vis spectroscopy from the decrease of the absorbance value of the supernatant and was found to be equal to 1.48 × 10⁻² mg DOX/mg Au.

Preparation and characterization of DOX-tethered AuNTs (AuNTs-PEG-DOX)

NIR – sensitive nanotriangles (AuNTs) were prepared by a rescaled version of the method reported by Zhang et al.⁶ Aqueous solutions of tetrachloroauric acid and sodium thiosulfate were mixed and stirred for approximately 3 minutes. 50 mL of 1.71 mM HAuCl₄·3H₂O was mixed with 15 mL of 3 mM Na₂S₂O₃ – until the colour changed from yellow to brown to dark violet. The solution was left for an hour until full stabilization of the AuNTs which was confirmed by UV-vis spectra indicating no further shift of the peak in the NIR region after 40 minutes, Fig. 3.

Fig. 3 UV-vis spectra of the stabilization process of AuNTs. (1) 3 minutes after mixing of substrates, (2) after 10 minutes, (3) after 20 minutes, (4–7) after 30–60 minutes.

The preparation of AuNTs-PEG-DOX was performed in the same way as for AuNPs-PEG-DOX, Scheme 2C. The final colloid solution of anisotropic nanoplates was separated from spherical gold NPs, which are also formed during this reaction, by centrifugation. Only a few spherical gold NPs remain after such a separation, Fig. 2D and 4. FE-SEM images obtained for AuNTs-PEG-DOX before and after separation are shown in Fig. 2C and D and confirm the decrease in concentration of small spherical AuNPs. A histogram of the AuNTs-PEG-DOX size distribution is shown in Fig. 2E. The concentration of doxorubicin on the AuNTs surface is equal to 1.72 × 10⁻² mg DOX/mg Au, as determined from the decrease in the absorbance of the doxorubicin supernatant after the conjugation and separation out of the pellet of AuNTs-PEG-DOX, compared with the control (the same synthesis but without AuNTs). The difference between the absorbance of the supernatant left after conjugation with DOX and the control solution allowed us to calculate the amount of DOX attached to the nanotriangles based on the Lambert–Beer equation (ESI†). The size of the synthesized AuNTs was obtained by DLS, Table 1. Due to the anisotropy of non-spherical NPs, the mean sizes obtained from DLS measurements may be overestimated.

Fig. 4 UV-vis spectra of AuNTs-PEG-DOX before separation of colloidal gold (1) and after first (2) (5000 rpm/10 min) and second (3) (4000 rpm/10 min) centrifugations, respectively.

Preparation of AuNPs loaded with DOX via pH-sensitive hydrazone bond (AuNPs-PEG-Hyd-DOX)

Fifty-nanometres citrate-capped AuNPs were prepared in the same way as for AuNPs-PEG-DOX. To get AuNPs-PEG-Hyd-DOX, 20 mL of an aqueous solution of as-prepared nanoparticles was modified with 4,7,10,13,16,19,22,25,32,35,38,41,44,47,50,53-hexadecaoxa-28,29-dithiahexapentacontanedioic acid di-N-succinimidyl ester – 0.8 mg of PEG NHS ester disulfide (n = 7) was dissolved in 1 mL of distilled water, added to the nanoparticles solution and stirred on a magnetic stirrer overnight. Next, the solution was centrifuged 5 times (5000 rpm, 45 min) in a special filtering flasks equipped with a dialysis membranes (5000 MWCO PES), Vivaspin 6, Sartorius Stedim Biotech, and rinsed with distilled water between each round to remove the excess cross-linker. In the next step, the solution was pre-concentrated to 100 μL. The concentrate was diluted with 5 mL of distilled water, and 20 μL of an aqueous solution of hydrazine monohydrate was added and the mixture was stirred overnight. During this process the colour changed from cherry-red to blood-red. Again, the solution was centrifuged as described above, concentrated to 100 μL and diluted with DMSO to 3 mL. The next step was the modification with DOX – 1.1 mg of DOX was dissolved in 0.2 mL of DMSO, added to the solution, and stirred for 24 h. The AuNPs-PEG-Hyd-DOX were cleaned 5-times by centrifuging, rinsed with water in between, and concentrated to finally obtain 500 μL of an aqueous solution of AuNPs-PEG-Hyd-DOX. The final product was stored in the refrigerator.

Drug release profiles monitored by fluorescence spectroscopy

While, like the majority of anthracycline drugs, free DOX is fluorescent, the formation of covalent bonds with gold nanoparticles quenches the fluorescence due to energy transfer from doxorubicin to the AuNPs. It has been reported that the fluorescence quenching efficiency in an NSET (nanosurface energy transfer) model depends on the distance between the donor and the quencher.²⁸ DOX conjugated with AuNPs is transferred to the cancer cells through endocytosis, and is initially present in endosomes, which have a pH in the range of 4.6 to 6.5, i.e., much lower than normal physiological conditions.²⁹

While the peptide bond is insensitive to pH changes, the hydrazone bond is pH-sensitive, and the drug is released from the carrier once encapsulated in the endosome, which is visible as the fluorescence recovery. Fig. 5 shows the profiles of DOX release from AuNPs-PEG-Hyd-DOX and AuNTs-PEG-DOX (same tendency for AuNPs-PEG-DOX) in solutions of pH 5.5 and 7.4. After 7 hours, almost 80% of the DOX was released at pH 5.5, while only 10% of the drug was released at pH 7.4. This result confirms that the release of DOX from AuNPs-PEG-Hyd-DOX in a more acidic environment was due to cleavage of the hydrazone bond. For AuNTs-PEG-DOX, the drug was not released at either pH 7.4 or 5.5 due to the high stability of the amide (peptide) bond in this range of pH. The same behaviour was encountered for AuNPs-PEG-DOX (results not shown).

Fig. 5 Monitoring of DOX release by fluorescence spectroscopy. AuNPs-PEG-Hyd-DOX (1,2) and AuNTs-PEG-DOX (3,4) in solutions of pH 5.5 (1,3) and 7.4 (2,4).

MTT assay of toxicities of AuNTs-PEG-DOX, AuNPs-PEG-DOX and AuNPs-PEG-Hyd-DOX

The MTT [3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide] test, which measures the activity of mitochondrial dehydrogenases, was used to evaluate the short-term cellular toxicity of the synthesized NSs. The assay was carried out as described elsewhere.³⁰ In brief, human epithelial lung carcinoma cell line A549 and human cervix carcinoma cell line HeLa were purchased from the American Type Tissue Culture Collection (ATCC, Rockville, MD, USA) and cultured in F12 or DMEM medium, respectively, according to the ATTC recommendations. The cells were plated at a density of 50 000 cells per well in 96-well plates and treated with the tested compounds at a concentration of 5 × 10⁻⁶ mol dm⁻³ or a 2.5 × 10⁻⁶ mol dm⁻³ or vehicle (control) for 24 or 48 h at 37 °C in a 5% CO₂ humidified atmosphere. After incubation, 10 μL of MTT (5 mg mL⁻¹ in PBS, pH 7) was added to each well, and the cells were incubated at 37 °C for 4 h in a humidified atmosphere. Then, the growth medium was removed, and 100 μL of DMSO was added to each well to dissolve the purple crystals of formazan. The absorbance was measured in a plate reader spectrophotometer (Infinite M200, Tecan, Morrisville, NC) at a wavelength of 570 nm. The cell metabolic activity, which roughly relates to the cell viability, was expressed as the ratio of the absorbance of the treated cells to the absorbance of the cells treated with the vehicle, both after the subtraction of the reagent control.

Due to mutation, human lung carcinoma A549 cells have an overstimulated xenobiotic stress response through the NRF/KEAP signalling pathway.³¹ A549 cells also show a strong response to the “survival-type” signalling pathways that makes them more resistant to apoptotic stimuli.³² All tested compounds showed time and concentration-dependent toxicity in both cell lines. In our previous work HeLa cells proved to be more sensitive to DOX than A549 cells.³³

As expected, HeLa cells also proved to be more sensitive to DOX than the A549 cells, especially when the higher concentration (5 × 10⁻⁶ M) was used. All synthesized gold-DOX conjugates were more toxic to both cell lines than DOX alone, regardless of the time of treatment. All Au-DOX conjugates synthesized in this study showed similar toxicities to both cell lines, despite the time of treatment or concentration, with the exception of the HeLa cells treated for 24 h with compounds at a higher concentration. All conjugates showed similar toxicities against the more sensitive HeLa cells. Interestingly, AuNTs-PEG-DOX and AuNPs-PEG-DOX were more toxic than DOX alone against the more resistant A549 cell line, (Fig. 6). On the other hand, neither gold nanospheres nor gold nanotriangles alone expressed any toxicity against either cell line tested.

Fig. 6 Toxicities of the synthesized carriers and DOX alone measured by the MTT assay on A549 (A) and HeLa (B) cancer cell lines. Statistical significance of the difference of means was tested by pair-wise comparison by Student's t-test for independent samples with a significance threshold p < 0.05. (1) denotes a significant difference between concentrations, (2) denotes a significant difference vs. DOX; (3) denotes a significant difference vs. triangles; (4) denotes a significant difference vs. spheres; (5) denotes a significant difference vs. time; (6) denotes a significant difference vs. another cell line.

Confocal microscopy

Due to the presence of an aromatic moiety, doxorubicin exhibits strong fluorescence that can be easily observed by confocal fluorescence microscopy. Indeed, the DOX fluorescence was clearly visible in the cytoplasm approximately 30 min after the start of the treatment. Interestingly, the DOX fluorescence inside the cells treated with the AuNTs- or AuNPs-PEG-DOX conjugate was noticeably weaker than that observed in the AuNPs-PEG-Hyd-DOX treated cells (Fig. 7, panels 1B, 2B, 3B), which confirmed the high release of DOX from NPs with pH-sensitive bonding.

Fig. 7 Confocal microscopy pictures of Hoechst-dyed nuclei (A column), DOX fluorescence (B column) and an overlay image (C column) obtained after 24 h for all three DOX carriers: AuNTs-PEG-DOX, AuNPs-PEG-DOX and AuNPs-PEG-Hyd-DOX. In 1C and 2C, for carriers with peptide bonds, the apoptotic pathway of death is visible. On the other hand, for the pH-active hydrazone bond, sudden necrotic death is present.

We also observed the visible hallmarks of DOX cytotoxicity, supporting the results of MTT test. A more detailed analysis revealed evidence of programmed long-term death (apoptosis) in cells treated with nanomaterials with the covalently bound drug, as indicated by cell shrinkage and blebbing (Fig. 7, panels 1B, 2B) and visible chromatin condensation (Fig. 7, panels 1A, 2A). The apoptosis process started approximately 7 h after nanomaterial addition and became significant after 24 h of exposure. On the other hand, cells treated with DOX connected through a pH-sensitive hydrazone bond exhibited evidence of a sudden, uncontrolled death (necrosis) rather than apoptosis (Fig. 7, panels 3A–C). The process started just 2 h after nanomaterial addition indicating that the possible release of the drug leads to the formation of some toxic species, most likely at the outer part of the detached carrier, Fig. 7. This hypothesis is further supported by our observation (data not shown) suggesting that the presence of free hydrazine groups on the carrier interfere with the MTT assay, reducing the dye in a cell-independent mode. Because we observed pronounced AuNPs-PEG-Hyd-DOX toxicity, the free hydrazine groups on the carrier end were not available or not accessible to the MTT dye.

Conclusions

Gold nanoparticles were prepared by two methods – the common Turkevich method and the thiosulfate method reported by Gobin et al.⁶ In the first case, spherical gold nanoparticles, AuNPs, of approximately 50 nm, were obtained, in the second, larger, approximately 70 nm, NIR-sensitive gold nanotriangles, AuNTs, were the product. The near-IR absorbing AuNTs are important because most biological tissue components show low absorption and high transmission of light in this wavelength region. Moreover, the NIR properties of AuNTs-PEG-DOX allow the multimodal action of such NPs as both thermal and chemotherapeutic agents. When doxorubicin is bound by means of a thiolated PEG linker, both types of nanoparticles were robust, stable and efficient, as indicated by the MTT assay and confocal microscopy studies. It should be underlined that both AuNPs-PEG-DOX and AuNTs-PEG-DOX exhibited increased cytotoxic efficiency compared to the free doxorubicin, as clearly shown on the example of the more resistant A549 cell line. The apoptosis process started approximately 7 h after nanomaterial addition and became significant after 24 h of exposure. Only slight differences in the AuNPs-PEG-DOX and AuNTs-PEG-DOX toxicities were noted, but the therapeutic effect of NIR-absorbing triangular nanostructures modified with DOX may be significantly enhanced by providing light of an appropriate wavelength. The induced hyperthermia of malignant cells would support anti-cancer therapy based on the application of anthracycline drugs.

The hydrazone bond used to modify AuNPs with DOX (AuNPs-PEG-Hyd-DOX) adds pH-sensitivity to the drug carrier and allows the control of the release of the drug from. Indeed, fluorescence spectroscopy measurements of AuNPs-PEG-Hyd-DOX revealed that the use of a hydrazone moiety as the linker led to a fast release of the drug at pH 5.5 (cancer cell environment), while almost no release was observed at pH 7.4 (normal cell environment), even after 20 h. On the other hand, cells treated with DOX connected through the pH-sensitive hydrazone bond exhibited the evidence of a sudden and uncontrolled death (necrosis) rather than apoptosis. The process started just 2 h after nanomaterial addition, indicating that the release of the drug leads to the formation of some toxic species at the outer part of the detached carrier. This observation was also supported by MTT data. Because necrotic cell death may lead to inflammatory processes and is disadvantageous compared with apoptotic death, these findings should be taken into account when choosing the linkers in the drug-carrier structures, e.g., in designing gold-nanoparticle carriers.

Acknowledgements

The project was financed by the National Science Centre conferred on the basis of decision number DEC-2014/13/B/ST5/04117.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra00177g

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