Synthesis of Bacteria-mimetic Gold Nanoparticles for Phagocytosis by Immune Cells

Materials and reagents

1. Falcon tube (Falcon, catalog number: 352098)

2. Millipore with 10 kDa filter (Millipore, catalog number: UFC901024)

3. Nylon membrane filter with 0.45 pore size (Sigma-Aldrich, catalog number: Z290815-100EA)

4. HAuCl₄·3H₂O (Sigma-Aldrich, catalog number: 520918)

5. Sodium citrate (Aladdin, catalog number: S189183-100g)

6. Mono(6-mercapto-6-deoxy)-beta-cyclodextrin (Shandong Binzhou Zhiyuan Biotechnology Co., Ltd, catalog number: 81644-55-5)

7. 1-adamantanethiol (Aladdin, catalog number: A169723-5g)

8. E. coli (ATCC, catalog number: 33694)

9. Polypropylene bacterial culture tube (Falcon, catalog number: 352057)

10. DMSO (Sigma-Aldrich, catalog number: D8418-100ML)

11. Lysogeny broth (LB) (Thermo Fisher Scientific, catalog number: 10855021)

12. LB medium (see Recipes)

13. Sodium citrate solution (5%) (see Recipes)

14. HAuCl₄ solution (0.25 mM) (see Recipes)

15. Mono(6-mercapto-6-deoxy)-beta-cyclodextrin solution (2 mM) (see Recipes)

16. GNP solution (10 mM) (see Recipes)

17. 1-adamantanethiol in DMSO (2 mM) (see Recipes)

Equipment

1. Centrifuge (Thermo Fisher Scientific, Heraeus Multifuge X3, model: 10325804)

2. Dynamic light scattering (Malvern, Nano-ZS)

3. Ultra-high-speed centrifuge (Beckman, Optima XPN-100 Ultracentrifuge)

4. Sonication bath (Branson, Bransonic ultrasonic cleaner, model: 5510E-DTH)

5. Transmission electron microscope, 120 kV (HITACHI, model: HT7800)

6. Microplate reader (Molecular Devices, SpectraMax, model: M5)

7. Vacuum oven (Thermo Fisher Scientific, Vacutherm, model: VT6205)

Software

1. Software associated with plate reader used (SoftMax Pro 7 Software)

2. Software associated with dynamic laser scanning used (Malvern Zetasizer Software v7.11)

3. Software associated with transmission electron microscope used (Gatan DigitalMicrograph 3.9)

Procedure

1. Preparation of GNPs via Turkevich method

   1. Use HAuCl₄in combination with sodium citrate as a reducing and capping agent for the nanoparticles.

   2. Prepare HAuCl₄ solution (0.25 mM; see Recipes) in a conical flask.

   3. Put 100 mL of HAuCl₄ solution (0.25 mM) into a 500 mL round-bottom flask with single neck and stir at 130 °C in an oil bath in a magnetic stirrer.

   4. When HAuCl₄ solution starts to boil, add 0.7 mL of sodium citrate solution (5%; see Recipes) into HauCl₄ solution.

   5. When the solution turns wine red, add 0.7 mL of sodium citrate solution (5%) again and repeat the addition process of sodium citrate solution twice.

   6. Cool the reaction solution down to room temperature and transfer the entire reaction mixture to multiple 2 mL polypropylene microcentrifuge tubes. Centrifuge at 12,000× g for 30 min to collected precipitated GNPs.

   7. Add 100 mL of ddH₂O to disperse the precipitated GNPs and centrifuge at 12,000× g for 30 min again. Repeat the washing twice with deionized water and collect purified GNPs.

   8. Characterize GNPs by transmission electron microscope (TEM) and dynamic light scattering (DLS). TEM imaging shows the morphology of Au nanoparticles and DLS analysis reflects the size distribution.

   9. Place the precipitated GNPs in a 2 mL polypropylene microcentrifuge tube and dry GNPs in the vacuum oven at 25 °C for two days (Figure 1a).

      
      

      
      Figure 1. Photos of different gold nanoparticles (GNP) precipitates. a) GNP solution and GNP precipitate. b) CD-GNP solution and CD-GNP precipitate. c) ADA-GNP solution and ADA-GNP precipitate. d) Isolated outer membrane vesicles (OMV) precipitate. e) OMV-coated CD-GNP solution and OMV-coated CD-GNP precipitate. f) OMV-coated ADA-GNP solution and OMV-coated ADA-GNP precipitate.

      
      



2. Preparation of CD-GNPs

   1. Put 1,000 mL of ddH₂O in a conical flask and add 2.3 g of mono(6-mercapto-6-deoxy)-beta-cyclodextrin to prepare mono(6-mercapto-6-deoxy)-beta-cyclodextrin solution (2 mM; see Recipes).

   2. Dry GNPs in a vacuum oven and dissolve 1.97 g of GNP power in 1,000 mL of ddH₂O to prepare GNPs solution (10 mM; see Recipes).

   3. Add 2 mL of mono(6-mercapto-6-deoxy)-beta-cyclodextrin solution (2 mM) to 2 mL of GNPs solution (10 mM) in a beaker and stir in a magnetic stirrer for 2 h at room temperature.

   4. Centrifuge the resultant solution at 21,000× g for 8 min in a 2 mL polypropylene microcentrifuge tube and collect the precipitated CD-GNPs.

   5. Dry CD-GNPs in a vacuum oven at 25 °C for two days (Figure 1b).




3. Preparation of ADA-GNPs

   1. Dissolve 1-adamantanethiol in DMSO (2 mM, 2 mL; see Recipes).

   2. Add GNP solution (10 mM, 2 mL) into 1-adamantanethiol solution in a beaker and stir for 2 h.

   3. Centrifuge the resultant solution at 21,000× g for 8 min in a polypropylene microcentrifuge tube and collect the precipitated ADA-GNPs.

   4. Dry ADA-GNPs in a vacuum oven at 25 °C for two days (Figure 1c).




4. Isolation of bacterial OMVs

   1. Inoculate E. coli in a polypropylene bacterial culture tube containing 50 mL of LB medium (see Recipes) for three days, and then centrifuge resulting bacterial culture in a 50 mL Falcon tube at 2,000× g for 5 min. Disperse the precipitated bacteria in ddH₂O to achieve a OD₆₀₀ value of 1.0, measured in a microplate reader at wavelength of 600 nm.

   2. Filter 50 mL of the supernatant through a 0.45 pore size nylon membrane filter.

   3. Transfer the resulting filtrate into Millipore 10 kDa filter and centrifuge at 5,000× g for 5 min to obtain the OMVs-rich solution in a Falcon tube.

   4. Centrifuge the OMVs-rich solution in a high-speed polypropylene copolymer (PPCO) centrifuge tube at ultra-high speed of 150,000× g for 3 h.

   5. Collect the precipitated OMVs in a polypropylene microcentrifuge tube and store at -80 °C (Figure 1d).




5. Synthesis of OMV-coated CD-GNPs

   1. Lyophilize OMVs to get OMV power.

   2. Mix 1 mg of OMVs and 1 mg of CD-GNPs in 50 mL of ddH₂O in Falcon tubes. Divide 50 mL of the resulting solution into two 50 mL Falcon tubes.

   3. Sonicate the Falcon tubes for 10 s at intervals of 10 s in a sonication bath at a frequency of 40 kHz (100 W) for 2 min at room temperature.

   4. Centrifuge the subsequent solution in the Falcon tube at 5,000× g for 30 min to precipitate OMV-coated CD-GNPs (Figure 1e).

   5. Place the solution of OMV-coated CD-GNPs in copper mesh and dry for TEM imaging, to analyze the coating layer on GNP surface.

   6. Place the solution of OMV-coated CD-GNPs in a quartz cuvette for DLS analysis, to measure the diameter change of GNP before and after OMV coating.




6. Synthesis of OMV-coated ADA-GNPs

   1. Mix 1 mg of OMVs and 1 mg of ADA-GNPs in 50 mL of ddH₂O solution. Divide 50 mL of the resulting solution into two 50 mL Falcon tubes.

   2. Sonicate Falcon tubes for 10 s at intervals of 10 s in a sonication bath at a frequency of 40 kHz (100 W) for 2 min at room temperature.

   3. Centrifuge the subsequent solution in the Falcon tube for 30 min at 5,000× g to precipitate OMV-coated ADA-GNPs (Figure 1f).

   4. Place the solution of OMV-coated ADA-GNPs in copper mesh and dry for TEM imaging, to analyze the coating layer on GNP surface.

   5. Place the solution of OMV-coated ADA-GNPs in quartz cuvette for DLS analysis, to measure the diameter change of GNP before and after OMV coating.

Data analysis

1. Normalize peak value of diameter curve to 1 (y-axis, corresponding to the frequency of occupied particle diameter).

2. TEM is a microscopy technique capable of imaging at a significantly higher resolution than light microscopes. This enables the instrument to capture fine details of nano-sized nanoparticles. GNP solution was added on ultra-thin carbon film and then analyzed by TEM. The appropriate view was chosen and imaged. Data file containing figure information was generated. Finally, this profile was loaded into Digital Micrograph and the TEM figure was obtained. The data analysis step is shown in Figure 2. Figure 3a and 3b show the morphology of GNP nanoparticles and GNP aggregates. DLS is a technique in physics that can be utilized to determine the size distribution profile of nano-sized particles in suspension or in solution. GNP solution was added into a glass cell and then analyzed by DLS. An Excel file with diameter distribution and corresponding intensity were generated. Finally, these data were loaded into GraphPad Prism with x-axis of diameter value and y-axis of intensity value, and the figure of size distribution was obtained. The photo of DLS instrument and data analysis step are shown in Figure 4. DLS shows a large size distribution of GNP aggregates in comparison to that of ADA-GNPs and CD-GNPs (Figure 3c).

   
   

   
   Figure 2. Data generation steps by TEM analysis

   
   

   
   Figure 3. Characterization of outer membrane vesicles (OMV)-coated gold nanoparticles (GNPs). a) TEM image of GNPs. Scale bar: 500 nm. b) TEM image of GNP aggregates. Scale bar: 2 μm. c) Size distribution of the mixture of CD-GNPs and ADA-GNPs determined by DLS. d) TEM image of M-GNPs. Scale bar: 50 nm. Insert: amplified M-GNPs (the scale bar was 20 nm). e) Size distribution of GNPs, M-GNPs, and OMVs determined by DLS. f) The protein bands of M-GNPs, OMVs, and E. coli in the gel by Coomassie Blue staining.

   
   

3. OMV-coated CD-GNPs/OMV-coated ADA GNPs were characterized by TEM and DLS. TEM was utilized to analyze the coating layer on GNP surface; a transparent membrane layer with ~6 nm thickness was observed on GNP surface (Figure 3d). DLS was utilized to measure the diameter change of GNP before and after OMV coating; an increased size from 30 to 45 nm after coating of GNP with OMV was observed (Figure 3e).

4. Fit the frequency-diameter using a X-Y model with GraphPad Prism 9.0 software (Figure 4c and 4e).

   
   

   
   Figure 4. Photo of DLS instrument and data generation steps

Recipes

1. HAuCl₄ solution (0.25 mM)

   HAuCl₄·3H₂O, 85 mg

   ddH₂O, 1,000 mL




2. Mono(6-mercapto-6-deoxy)-beta-cyclodextrin solution (2 mM)

   Mono(6-mercapto-6-deoxy)-beta-cyclodextrin, 2.3 g

   ddH₂O, 1,000 mL




3. GNP solution (10 mM)

   GNP, 1.97 g

   ddH₂O, 1,000 mL




4. 1-adamantanethiol in DMSO (2 mM)

   1-adamantanethiol, 336 mg

   
   

   DMSO, 1,000 mL




5. LB medium

   Tryptone, 10 g/L

   Yeast extract, 5 g/L

   NaCl, 10 g/L




6. Sodium citrate solution (5%)

   Sodium citrate, 50 g

   ddH₂O, 1,000 mL

Acknowledgments

This research was funded by Dr. Stanley Ho Medical Development Foundation (Grant No.: SHMDF-OIRFS/2021/002), Science and Technology Development Fund (FDCT) Macau SAR (Grant No.: 0065/2021/A2), and National Natural Science Foundation of China (22071275).

The current protocol is derived from the original paper (Gao et al., 2022).

Competing interests

R.W. and C.G. have one pending patent based on the bacteria-mimetic gold nanoparticles presented in this manuscript.

References

1. Gao, C., Wang, Q., Li, J., Kwong, C. H. T., Wei, J., Xie, B., Lu, S., Lee, S. M. Y. and Wang, R. (2022). In vivo hitchhiking of immune cells by intracellular self-assembly of bacteria-mimetic nanomedicine for targeted therapy of melanoma. Sci Adv 8(19): eabn1805.
2. Krueger, T. E. G., Thorek, D. L. J., Denmeade, S. R., Isaacs, J. T. and Brennen, W. N. (2018). Concise Review: Mesenchymal Stem Cell-Based Drug Delivery: The Good, the Bad, the Ugly, and the Promise. Stem Cells Transl Med 7(9): 651-663.
3. Li, T., Dong, H., Zhang, C. and Mo, R. (2018). Cell-based drug delivery systems for biomedical applications. Nano Res 11(10): 5240-5257.
4. Li, Z., Wang, Y., Ding, Y., Repp, L., Kwon, G. S. and Hu, Q. (2021). Cell-Based Delivery Systems: Emerging Carriers for Immunotherapy. Adv Funct Mater 31(23): 2100088.
5. Loescher S. and Walther A. (2020). Supracolloidal Self‐Assembly of Divalent Janus 3D DNA Origami via Programmable Multivalent Host/Guest Interactions. Angew Chem Int Engl 59 (14): 5515-5520.
6. Wang, H., Wu, H., Yi, Y., Xue, K-F., Xu, J-F., Wang, H., Zhao, Y. and Zhang, X. (2021). Self-Motivated Supramolecular Combination Chemotherapy for Overcoming Drug Resistance Based on Acid-Activated Competition of Host–Guest Interactions. CCS Chemistry 3(8): 1413-1425.
7. Yang, L., Yang, Y., Chen, Y., Xu, Y. and Peng, J. (2022). Cell-based drug delivery systems and their in vivo fate. Adv Drug Deliv Rev 187: 114394.
8. Zhang, J., Yang, L., Huang, F., Zhao, C., Liu, J., Zhang, Y., and Liu, J. (2021). Multifunctional hybrid hydrogel enhanced antitumor therapy through multiple destroying DNA functions by a triple‐combination synergistic therapy. Adv Healthc Mater, 10(21): 2101190.