Nanoparticle Sterilization Strategies in plant tissue culture

Sterilization of nanoparticles (NPs) is a crucial step before their application in plant tissue culture, including elicitation for secondary metabolite production. The sterilization method should ensure that the nanoparticles remain biologically active and free from contaminants while maintaining their physicochemical properties. Here are some commonly used sterilization techniques for nanoparticles in elicitation studies:

1. Autoclaving (High-Pressure Steam Sterilization)

Autoclaving is a widely used sterilization method that employs high-pressure steam at 121°C and 15 psi for 15–20 minutes to eliminate microbial contamination. This method is suitable for heat-stable materials, including some types of nanoparticles.

Mechanism of Action

1. High temperature (121°C) and pressure (15 psi) destroy bacterial spores, fungi, and viruses.
2. Steam penetrates the nanoparticle suspension, ensuring thorough sterilization.
3. Moist heat coagulates microbial proteins, leading to complete sterilization.

Nanoparticles Suitable for Autoclaving

• Metal Oxide NPs: Zinc oxide (ZnO), Titanium dioxide (TiO₂), Iron oxide (Fe₃O₄).
• Silica-Based NPs: Silica nanoparticles (SiO₂), Alumina (Al₂O₃).
• Carbon-Based NPs: Carbon nanotubes (CNTs), Graphene oxide (GO) (depending on dispersion medium).

Nanoparticles NOT Suitable for Autoclaving

• Silver (Ag) and Gold (Au) Nanoparticles → May undergo aggregation or oxidation.
• Polymeric Nanoparticles (PLGA, Chitosan, Alginate, Liposomes, etc.) → Can degrade at high temperatures.
• Lipid-Based Nanoparticles → Heat can disrupt lipid structures.

Steps for Autoclaving Nanoparticle Suspension

• Prepare NP Suspension: Dispense in heat-resistant glass or autoclavable plastic bottles.
• Loosely Cap the Container: Prevent pressure buildup.
• Autoclave at 121°C, 15 psi for 15–20 min.
• Cool to Room Temperature: Allow NPs to settle before further use.
• Check for Aggregation: Use UV-Vis spectroscopy or DLS (Dynamic Light Scattering) to ensure stability.

Advantages of Autoclaving for Nanoparticle Sterilization

✔ Highly effective in sterilization.

✔ Cost-effective and widely available.

✔ Simple, does not require specialized chemicals.

Disadvantages

✖ May cause aggregation or size modification of nanoparticles.

✖ Not suitable for heat-sensitive nanoparticles.

✖ Possible surface oxidation for metallic NPs.

2. Filtration (Membrane Filtration)

Membrane filtration is a sterilization method that physically removes microorganisms from nanoparticle (NP) suspensions by passing them through a sterile membrane filter (0.22 µm or 0.45 µm pore size). This method is suitable for heat-sensitive nanoparticles that cannot withstand autoclaving or radiation.

Mechanism of Action

• Physical Separation: Bacteria and fungi are larger than 0.22 µm, so they are trapped by the membrane, allowing only nanoparticle suspension to pass through.
• Preserves NP Integrity: No heat or radiation is involved, preventing aggregation or degradation.
• Maintains Sterility: The filtered solution remains sterile if handled in aseptic conditions.

Nanoparticles Suitable for Filtration

• Polymeric NPs: PLGA, Chitosan, Alginate, Liposomes.
• Lipid-Based NPs: Nanoemulsions, Liposomes, Solid Lipid Nanoparticles (SLNs).
• Small Metallic NPs: Silver (Ag), Gold (Au), Copper (Cu), if well dispersed.
• Quantum Dots: CdSe, ZnS, or similar nanomaterials.

Nanoparticles NOT Suitable for Filtration

❌ Agglomerated or Large-Sized NPs (>0.22 µm) → May clog the membrane.

❌ Highly Viscous Suspensions → Difficult to pass through the filter.

❌ Magnetic NPs (Fe₃O₄, etc.) → Can stick to filter material.

❌ Insoluble or Poorly Dispersed NPs → May block the filter.

Steps for Membrane Filtration

• Prepare NP Suspension: Ensure the nanoparticles are well dispersed in a sterile buffer or medium.
• Choose the Right Filter: Use a 0.22 µm membrane filter (for complete sterilization) or 0.45 µm (if clogging occurs)

• Use a Sterile Syringe or Vacuum Filtration Unit:

  • Syringe Filtration: Attach a sterile filter to a syringe and slowly push the suspension through.

  • Vacuum Filtration: Use a vacuum pump with a sterile filtration assembly for larger volumes.

• Collect Sterile Nanoparticle Suspension: Store in a sterile container under aseptic conditions.

Advantages of Membrane Filtration for NP Sterilization

✔ Maintains Nanoparticle Stability – No heat or radiation exposure.

✔ Preserves Surface Functionalization – No chemical modifications.

✔ Fast and Simple – Immediate sterilization without special equipment.

✔ Ideal for Heat-Sensitive and Biodegradable NPs (e.g., polymeric or lipid-based NPs).

Disadvantages

✖ Not Suitable for Large-Sized or Agglomerated NPs.

✖ Filter Clogging – Requires pre-dilution or sonication.

✖ Not Effective Against Viruses or Endotoxins – Only removes bacteria and fungi.

3. UV Sterilization

UV sterilization is a non-thermal, chemical-free method that uses ultraviolet (UV-C) light (typically 254 nm wavelength) to eliminate microbial contamination in nanoparticle (NP) suspensions. It is suitable for heat-sensitive nanoparticles that cannot withstand autoclaving or radiation.

Mechanism of Action

• DNA Damage: UV light disrupts microbial DNA, preventing replication and leading to cell death.
• Surface Decontamination: Kills bacteria, fungi, and some viruses present in NP suspensions.
• Minimal NP Alteration: Unlike heat or chemicals, UV does not significantly change NP properties.

Nanoparticles Suitable for UV Sterilization

Metallic NPs: Silver (Ag), Gold (Au), Copper (Cu).

Polymeric NPs: PLGA, Chitosan, Alginate (if in suspension).

Silica-Based NPs: SiO₂, Alumina (Al₂O₃).

Quantum Dots: CdSe, ZnS.

Nanoparticles NOT Suitable for UV Sterilization

❌ Opaque or Highly Absorptive NPs (e.g., Carbon Nanotubes, Graphene Oxide) → UV cannot                    penetrate.

❌ Highly Concentrated or Turbid Suspensions → Blocks UV penetration.

❌ Magnetic NPs (Fe₃O₄, etc.) → UV may be less effective if NPs aggregate.

❌ Large Volumes (>10 mL) → UV is less effective for bulk sterilization.

Steps for UV Sterilization

• Prepare NP Suspension: Ensure it is well dispersed in a sterile container.
• Use a UV Chamber or UV Lamp: Place the container 5–10 cm away from a 254 nm UV-C source.
• Expose for 30–60 Minutes: Stir the suspension every 10 minutes for even exposure.
• Check NP Stability: Perform DLS, UV-Vis, or SEM analysis to confirm no aggregation.

Advantages of UV Sterilization for Nanoparticles

✔ Preserves NP Structure – No heat or chemicals involved.

✔ Quick and Cost-Effective – Takes 30–60 min, requires only a UV lamp.

✔ Suitable for Heat-Sensitive Nanoparticles – Ideal for polymeric and organic NPs.

Disadvantages

✖ Not Effective for Opaque or Highly Concentrated Suspensions.

✖ Does Not Remove Endotoxins or Small Viral Particles.

✖ Requires Aseptic Handling After Sterilization to prevent recontamination..

4. Gamma Irradiation

Gamma irradiation is a high-energy, non-contact sterilization method that uses gamma rays (typically from Cobalt-60 or Cesium-137) to eliminate microbial contamination in nanoparticles (NPs). It is particularly useful for bulk sterilization and heat-sensitive nanoparticles that cannot withstand autoclaving.

Mechanism of Action

• Ionizing Radiation: Gamma rays generate reactive species (e.g., free radicals) that break microbial DNA, proteins, and cell membranes.
• Deep Penetration: Unlike UV, gamma rays can sterilize thick suspensions and solid nanoparticle powders.
• Preserves Sterility: Once sealed in sterile packaging, gamma-irradiated NPs remain sterile.

Nanoparticles Suitable for Gamma Irradiation

• Metallic NPs: Silver (Ag), Gold (Au), Zinc Oxide (ZnO), Iron Oxide (Fe₃O₄), Titanium Dioxide (TiO₂).
• Polymeric NPs: PLGA, Chitosan, Alginate (if properly stabilized).
• Silica-Based NPs: SiO₂, Alumina (Al₂O₃).
• Carbon-Based NPs: Graphene, Carbon Nanotubes (CNTs).
• Quantum Dots: CdSe, ZnS.

Nanoparticles NOT Suitable for Gamma Irradiation

❌ Highly Reactive or Degradable NPs (e.g., Some Lipid-Based NPs) → May degrade under irradiation.

❌ NPs Containing Organic Ligands or Coatings → Surface modifications may be altered.

❌ Protein-Conjugated NPs → Protein structures may be disrupted.

Steps for Gamma Irradiation

1. Prepare NP Suspension or Powder: Dispense in sterile, sealed vials or vacuum-sealed bags.

2. Choose an Appropriate Radiation Dose:

   • 10–25 kGy → Effective sterilization without altering most NP properties.

   • Higher doses (>25 kGy) → May affect polymeric NPs.

3. Expose to Gamma Rays (Cobalt-60 Source): Typically done in a specialized irradiation facility.

4. Analyze NP Properties Post-Irradiation: Check for changes in size, zeta potential, and surface chemistry.

Advantages of Gamma Irradiation for Nanoparticle Sterilization

✔ Highly Effective – Kills bacteria, fungi, and spores, even in dense suspensions.

✔ No Heat Required – Suitable for heat-sensitive nanoparticles.

✔ Suitable for Bulk Sterilization – Works for large NP batches.

✔ Penetrates Deeply – Effective for thick samples or powder forms.

Disadvantages

✖ Requires Specialized Equipment – Must be done in a gamma irradiation facility.

✖ Potential NP Modifications – Some surface coatings or polymeric NPs may degrade.

✖ Expensive for Small-Scale Use – Costly compared to UV or filtration.

5. Ethanol Treatment

Ethanol treatment is a chemical sterilization method that uses 70% ethanol (EtOH) to eliminate microbial contamination in nanoparticle (NP) suspensions or powders. It is particularly useful for metallic and heat-sensitive nanoparticles that cannot withstand autoclaving or gamma irradiation.

Mechanism of Action

• Protein Denaturation: Ethanol disrupts microbial proteins and membranes, killing bacteria, fungi, and viruses.
• Dehydration: Ethanol dehydrates cells, leading to microbial death.
• Non-Thermal Sterilization: Unlike heat-based methods, ethanol does not alter NP stability significantly.

Nanoparticles Suitable for Ethanol Sterilization

• Metallic NPs: Silver (Ag), Gold (Au), Copper (Cu), Zinc Oxide (ZnO).
• Silica-Based NPs: SiO₂, Alumina (Al₂O₃).
• Carbon-Based NPs: Carbon Nanotubes (CNTs), Graphene.
• Polymeric NPs: PLGA, Chitosan, Alginate (if properly washed afterward).

Nanoparticles NOT Suitable for Ethanol Sterilization

❌ Lipid-Based NPs → Ethanol can dissolve or degrade liposomes and emulsions.

❌ Highly Hydrophobic NPs → May not disperse well in ethanol.

❌ Reactive NPs (e.g., Fe₃O₄) → Ethanol may alter surface chemistry.

Steps for Ethanol Sterilization

1. Prepare NP Suspension or Powder: Dispense in a sterile container.

2. Add 70% Ethanol: Ensure complete NP submersion.

3. Incubate for 15–30 Minutes: Stir occasionally for even exposure.

4. Remove Ethanol:

   • For Suspensions: Centrifuge and wash with sterile water or PBS (3–5 times).

   • For Powders: Air-dry under sterile conditions.

5. Confirm Sterility: Use microbial culture tests if necessary.

Advantages of Ethanol Sterilization for Nanoparticles

✔ Preserves NP Stability – No heat or radiation.

✔ Simple and Cost-Effective – Requires only ethanol and washing steps.

✔ Fast Sterilization – Takes only 15–30 minutes.

Disadvantages

✖ Requires Thorough Washing – Residual ethanol may interfere with biological applications.

✖ Not Suitable for Lipid-Based NPs – Ethanol can disrupt lipid structures.

✖ Not Effective Against Endospores – May require combination with filtration or UV.

6. Plasma Sterilization

Plasma sterilization is an advanced, non-thermal method that uses ionized gas (plasma) containing reactive oxygen and nitrogen species (ROS & RNS) to eliminate microbial contamination in nanoparticles (NPs). This method is ideal for heat-sensitive and delicate nanoparticles that may degrade under traditional sterilization techniques.

Mechanism of Action

• Reactive Species Attack Microbial Cells: Plasma generates free radicals (O·, OH·, NO·) that damage microbial DNA and proteins.
• Oxidation & Etching Effect: Disrupts microbial membranes without affecting nanoparticle integrity.
• Non-Thermal Process: Unlike autoclaving or gamma irradiation, plasma sterilization occurs at low temperatures (~30–50°C).

Nanoparticles Suitable for Plasma Sterilization

• Metallic NPs: Silver (Ag), Gold (Au), Copper (Cu), Titanium Dioxide (TiO₂), Zinc Oxide (ZnO).
• Polymeric NPs: PLGA, Chitosan, Alginate (if stable).
• Carbon-Based NPs: Carbon Nanotubes (CNTs), Graphene, Fullerenes.
• Quantum Dots: CdSe, ZnS.
• Silica-Based NPs: SiO₂, Alumina (Al₂O₃).

Nanoparticles NOT Suitable for Plasma Sterilization

❌ Highly Reactive NPs → Some polymeric or lipid-based NPs may be altered.

❌ NPs with Organic Surface Modifications → Plasma may oxidize functional groups, changing NP properties.

❌ Volatile or Highly Soluble NPs → May be lost due to plasma-induced desorption.

Steps for Plasma Sterilization

1. Prepare NP Sample: Place the NP powder or suspension in a sterile, plasma-compatible container.

2. Expose to Plasma: Use low-temperature gas plasma (O₂, H₂O₂, or Argon-based plasma) inside a plasma chamber.

3. Set Exposure Time (15–60 min): Adjust based on NP type and microbial load.

4. Post-Sterilization Analysis: Check for size, zeta potential, and functional group integrity using DLS, FTIR, or UV-Vis spectroscopy.

Advantages of Plasma Sterilization for Nanoparticles

✔ Non-Thermal Process – Preserves NP structure.

✔ No Chemical Residues – Unlike ethanol or gamma sterilization.

✔ Effective for Heat-Sensitive & Delicate NPs.

✔ Fast & Efficient – Sterilization in 15–60 minutes.

Disadvantages

✖ Expensive & Requires Specialized Equipment.

✖ Potential Surface Modifications – Plasma may alter NP coatings or functional groups.

✖ Not Suitable for Some Polymeric or Lipid-Based NPs – May degrade under plasma exposure.

Sterilization Method	Mechanism of Action	Suitable Nanoparticles	Not Suitable Nanoparticles	Advantages	Disadvantages
Autoclaving (121°C, 15 psi)	High-temperature steam destroys microbes	Metal oxides (ZnO, TiO₂, Fe₃O₄), Silica (SiO₂, Al₂O₃), Some carbon-based NPs	Silver (Ag), Gold (Au), Polymeric (PLGA, Chitosan), Lipid-based NPs	✔ Highly effective ✔ Cost-effective ✔ Simple to use	✖ May cause aggregation or oxidation                     ✖ Not suitable for heat-sensitive NPs
Membrane Filtration (0.22 µm filter)	Physical removal of microbes	Polymeric (PLGA, Chitosan), Lipid-based, Well-dispersed small metallic NPs (Ag, Au), Quantum dots	Large-sized NPs, Agglomerated NPs, Magnetic NPs (Fe₃O₄)	✔ Maintains NP stability ✔ No heat or radiation ✔ Ideal for biodegradable NPs	✖ Not suitable for large/agglomerated NPs                             ✖ Filter clogging issues
UV Sterilization (254 nm UV-C)	Disrupts microbial DNA	Metallic (Ag, Au, Cu), Polymeric (PLGA, Chitosan), Silica (SiO₂), Quantum dots	Opaque/turbid suspensions, Carbon-based NPs (CNTs, Graphene), Magnetic NPs	✔ No heat/chemicals ✔ Quick and cost-effective ✔ Preserves NP properties	✖ Ineffective for opaque or concentrated suspensions                 ✖ Does not remove endotoxins
Gamma Irradiation (Cobalt-60, Cesium-137)	Ionizing radiation breaks DNA & proteins	Metallic (Ag, Au, ZnO, TiO₂, Fe₃O₄), Silica (SiO₂), Carbon-based, Quantum dots	Lipid-based NPs, NPs with organic ligands or proteins	✔ Deep penetration ✔ No heat ✔ Suitable for bulk sterilization	✖ Requires specialized facilities                      ✖ May alter polymeric NPs
Ethanol Treatment (70% ethanol)	Protein denaturation & dehydration	Metallic (Ag, Au, Cu, ZnO), Silica (SiO₂), Carbon-based	Lipid-based NPs, Hydrophobic NPs, Reactive NPs (Fe₃O₄)	✔ Simple & cost-effective ✔ No heat or radiation ✔ Fast sterilization	✖ Requires thorough washing                       ✖ Not effective against endospores
Plasma Sterilization (Ionized gas plasma)	Free radicals disrupt microbial structures	Metallic (Ag, Au, Cu, ZnO, TiO₂), Polymeric (PLGA, Chitosan), Carbon-based, Quantum dots, Silica (SiO₂)	NPs with organic coatings, Highly reactive NPs, Lipid-based NPs	✔ No heat/chemical residues ✔ Preserves NP structure ✔ Fast (15-60 min)	✖ Expensive equipment required     ✖ Possible surface modifications