Callus Culture: A Comprehensive Overview of Induction, Sub culturing, Classification, and Biotechnological Applications

Callus Culture

Callus culture is a fundamental technique in plant tissue culture, involving the growth of undifferentiated plant cell masses in a controlled, artificial environment. Here's a more in-depth look:

What is Callus?

A callus is an unorganized, proliferating mass of parenchyma cells. It forms when plant tissues are subjected to certain stimuli, particularly in response to wounding or when exposed to specific plant growth regulators. In essence, it's a mass of plant cells that have "dedifferentiated," meaning they've lost their specialized functions and reverted to a more basic, dividing state.

The Callus Culture Process:

1. Explant Selection:
• The process begins with selecting a suitable plant tissue, known as an explant. This can be a piece of leaf, stem, root, or other plant part.
2. Sterilization:
• Strict sterilization is crucial to prevent contamination by microorganisms. The explant is thoroughly sterilized to eliminate any bacteria or fungi.
3. Culture Medium:
• The explant is placed on a nutrient-rich culture medium. This medium typically contains:

• Essential mineral salts.
• Vitamins.
• Sugars (as an energy source).
• Plant growth regulators (hormones), such as auxins and cytokinins, which are critical for inducing callus formation.

4. Incubation:
• The cultures are incubated under controlled environmental conditions, including temperature, light, and humidity.
5. Callus Formation:
• Over time, the explant cells begin to divide and form a callus.
6. Subculturing:
• To maintain the callus culture, portions of the callus are periodically transferred to fresh culture medium (subcultured). This provides a continuous supply of nutrients and prevents the accumulation of waste products.
7. Regeneration (Optional):
• Depending on the desired outcome, the callus can be induced to regenerate whole plants through:

• Organogenesis: The formation of organs (shoots and roots) from the callus 
• Somatic embryogenesis: The formation of embryos from the callus cells.

Applications of Callus Culture:

• Micropropagation: Rapidly producing large numbers of genetically identical plants.
• Genetic engineering: Introducing foreign genes into plant cells.
• Production of secondary metabolites: Obtaining valuable compounds (e.g., pharmaceuticals) from plant cells.
• Plant breeding: Creating new plant varieties.
• Germplasm preservation: Conserving rare or endangered plant species.

Subculture of Callus

Subculturing refers to the transfer of callus from an old medium to a fresh medium after a certain time (typically 3–4 weeks), to maintain active growth and prevent browning or senescence. It helps in:

Purpose of Subculturing:

• Replenishing Nutrients:
• As callus grows, it depletes the nutrients in the culture medium. Subculturing involves transferring the callus to a fresh medium, ensuring a continuous supply of essential nutrients for healthy growth.
• Preventing Accumulation of Waste Products:
• Metabolic byproducts can accumulate in the culture medium, potentially inhibiting callus growth or causing toxicity. Subculturing helps to remove these waste products.
• Maintaining Callus Viability:
• Regular subculturing helps to maintain the viability and vigor of the callus culture, preventing it from senescence or death.
• Promoting Continued Growth:
• By providing fresh medium, subculturing stimulates continued cell division and proliferation, ensuring a consistent supply of callus tissue.
• Controlling Callus Characteristics:
• Sub culturing can also be used to influence the characteristics of the callus, for example by changing the hormone composition of the media.

The Sub culturing Process:

1. Preparation:

• A fresh batch of sterile culture medium is prepared.
• Sterile tools and a laminar flow hood are used to maintain aseptic conditions.

2. Callus Transfer:

•  A portion of the actively growing callus is carefully excised from the existing culture.
• This callus tissue is then transferred to the fresh culture medium.

3. Incubation:
• The subcultured callus is incubated under controlled environmental conditions, such as temperature, light, and humidity.
4. Frequency:
• The frequency of subculturing depends on the growth rate of the callus and the specific requirements of the plant species.

• Maintaining the viability of callus 
• Enhancing biomass
• Inducing differentiation (if required)
• Avoiding nutrient depletion and accumulation of toxic metabolites

Types of Callus Based on Texture and Color

1. Friable Callus

In plant tissue culture, "friable callus" refers to a specific texture of callus tissue. Here's a breakdown:

Callus:

A callus is an undifferentiated mass of plant cells. It forms in response to wounding or when plant tissue (an explant) is placed on a culture medium containing plant growth regulators.

Friable Callus:

• This type of callus is characterized by its loose, crumbly, and easily separable texture. The cells are loosely attached and have a relatively high-water content.
•  In contrast to a "compact callus," which is dense and firm, a friable callus is soft and delicate.
•  Friable callus is often desirable for certain applications in plant biotechnology, particularly for:
• Cell suspension cultures: Friable callus can be easily broken up and transferred to liquid media for cell suspension cultures, which are used for producing secondary metabolites or for genetic transformation.
• Somatic embryogenesis: Some friable callus types are more conducive to the development of somatic embryos, which can then be grown into whole plants.
• Loose, crumbly, and soft texture
• Useful for suspension culture
• Usually light in color (whitish or yellowish)

2. Compact Callus

When discussing callus culture in plant tissue culture, "compact callus" refers to a specific type of callus with distinct characteristics. Here's a breakdown:

Characteristics of Compact Callus:

• Dense and Firm:
• Unlike friable callus, which is loose and crumbly, compact callus is characterized by its tightly packed cells, resulting in a firm and dense texture.
• Tightly Aggregated Cells:
• The cells within a compact callus are closely adhered to one another, contributing to its solid appearance.
• Often Green:
• Compact callus may often have a greenish coloration, especially if it's derived from tissues with chloroplasts.
• Relatively Dry:
• Compared to a friable callus, a compact callus will usually contain less water.

Contrasting with Friable Callus:

• It's important to differentiate compact callus from friable callus. Friable callus has a loose, crumbly texture, making it more suitable for cell suspension cultures.
• Compact callus is denser.
• The texture of the callus is very dependent on the hormone balances within the growth medium.

Factors Influencing Callus Type:

• Plant Growth Regulators:
• The ratio of auxins to cytokinins in the culture medium significantly influences callus texture. Different ratios can favor the formation of either compact or friable callus.
• Plant Species and Genotype:
• Different plant species and even different cultivars within a species exhibit variations in callus texture.
• Culture Conditions:
• Environmental factors like light, temperature, and the composition of the basal medium also play a role.

Significance:

•  While friable callus is often preferred for cell suspension cultures, compact callus can be suitable for other applications in plant tissue culture.
• It is a form of callus that is produced, and then can be manipulated to produce different plant parts, through the changing of the hormone balances in the growth medium.
• Hard, dense, and tightly packed cells
• Often greenish or cream-colored
•  Less suitable for suspension cultures

3. Embryogenic Callus

Embryogenic callus is a specialized form of callus that holds significant importance in plant tissue culture. Here's a detailed explanation:

Key Characteristics:

• Potential for Somatic Embryogenesis:
• The defining characteristic of embryogenic callus is its ability to give rise to somatic embryos. Somatic embryos are embryos that develop from plant cells other than zygotes (fertilized eggs).
• Organized Structure:
• Unlike undifferentiated callus, embryogenic callus often exhibits a degree of organization, with cells that are predisposed to develop into embryos.
• Distinct Cellular Features:
• Embryogenic cells tend to be small, densely cytoplasmic, and have prominent nuclei.
• High Regeneration Capacity:
• This type of callus has a high capacity for regeneration, meaning it can efficiently produce whole plants.

Significance in Plant Tissue Culture:

• Efficient Plant Regeneration:
• Embryogenic callus is highly valuable for regenerating large numbers of plants, especially in species that are difficult to propagate through conventional methods.
• Genetic Transformation:
• It serves as an excellent target for genetic transformation techniques, allowing for the introduction of desired genes into plants. The resulting somatic embryos can then develop into transgenic plants.
• Clonal Propagation:
• Embryogenic callus allows for the clonal propagation of plants, ensuring that all regenerated plants are genetically identical to the parent plant.
• Plant Breeding:
• It's utilized in plant breeding programs to generate new plant varieties.

Factors Influencing Embryogenic Callus Formation:

• Plant Growth Regulators:
• The type and concentration of plant growth regulators, particularly auxins, play a crucial role in inducing embryogenic callus formation.
• Genotype:
• The genetic makeup of the plant species or cultivar significantly influences its ability to produce embryogenic callus.
• Explant Source:
• The type of explant used (e.g., leaf, stem, root) can affect the formation of embryogenic callus.
• Culture Medium:
• The composition of the culture medium, including nutrients and other additives, is essential for optimal embryogenic callus development.
• Capable of forming somatic embryos
• Usually yellowish-white or translucent
• Totipotent and can regenerate into whole plants

4. Non-embryogenic Callus

"Non-embryogenic callus" refers to callus tissue that lacks the capacity to produce somatic embryos. This distinguishes it from embryogenic callus, which is specifically characterized by its ability to develop into embryos. Here's a breakdown:

Key Characteristics:

• Lack of Somatic Embryogenesis:
• The defining feature of non-embryogenic callus is its inability to form somatic embryos, even when exposed to conditions that would induce embryogenesis in suitable callus tissue.
• Variable Morphology:
• Non-embryogenic callus can exhibit a range of textures, including friable (loose and crumbly) or compact (dense and firm).
• Different Cellular Structure:
• Compared to embryogenic callus, which often has cells with dense cytoplasm and prominent nuclei, non-embryogenic callus may have cells with larger vacuoles and a less organized structure.
• Different biochemical properties:
• There are differences in the protein and enzyme production in non-embryogenic callus, when compared to embryogenic callus.

Significance:

• Understanding Developmental Pathways:
• Studying non-embryogenic callus helps researchers understand the factors that control somatic embryogenesis. By comparing it with embryogenic callus, they can identify the genes and proteins involved in embryo development.
• Optimizing Tissue Culture Protocols:
• Understanding the conditions that lead to non-embryogenic callus formation is essential for optimizing tissue culture protocols and maximizing the production of embryogenic callus.
• Basic research:
• It is used in many basic research projects, to better understand plant cellular biology.
• Cannot form somatic embryos
• Used mainly for metabolite production or transformation

5. Organogenic Callus

Organogenic callus is a type of callus in plant tissue culture that has the capacity to develop into organized plant organs, such as shoots or roots. This is a key distinction from undifferentiated callus, which lacks this organizational potential. Here's a more detailed explanation:

Key Characteristics:

• Organ Formation:
• The defining characteristic is the ability to differentiate into specific plant organs. This process, known as organogenesis, can lead to the formation of shoots, roots, or even flowers.
• Organized Development:
• Unlike the random cell proliferation in undifferentiated callus, organogenic callus exhibits a degree of organized development, with cells forming distinct structures.
• Response to Hormones:
• The development of organs from organogenic callus is heavily influenced by the balance of plant growth regulators, particularly auxins and cytokinins, in the culture medium.
• Potential for Plant Regeneration:
• Organogenic callus is a valuable source for regenerating whole plants, particularly through the formation of shoots and roots.

Significance in Plant Tissue Culture:

• Plant Regeneration:
• It is a crucial tool for regenerating plants, especially in species that are difficult to propagate through other means.
• Micropropagation:
• Organogenic callus is used in micropropagation to produce large numbers of genetically identical plants.
• Genetic Transformation:
• It can serve as a target for genetic transformation, allowing for the introduction of desired genes into plants.
• Plant Breeding:
• It plays a role in plant breeding programs, facilitating the development of new plant varieties.
• Has potential to form organs like roots or shoots
• Often forms in response to specific PGR ratios

6. Pigmented Callus

Pigmented callus refers to callus tissue in plant tissue culture that exhibits coloration due to the production of various pigments. This coloration can vary significantly depending on the plant species, the specific pigments produced, and the culture conditions. Here's a more detailed explanation:

Causes of Pigmentation:

• Secondary Metabolites:
• Many plant species produce secondary metabolites, such as flavonoids, anthocyanins, carotenoids, and betalains, which can impart color to the callus.
• These compounds often serve protective functions in plants, such as UV protection or defense against pathogens.
• Chlorophyll:
• If the callus develops from tissues containing chloroplasts (e.g., leaves), it may exhibit a green coloration due to the presence of chlorophyll.
• Accumulation of Compounds:
• Sometimes the plant will accumulate different compounds within the callus, in response to the growth medium, or environmental conditions in the growth chamber.

Significance:

• Production of Valuable Compounds:
• Pigmented callus can be a valuable source for producing commercially important pigments or other secondary metabolites.
• For example, callus cultures of certain plants can be used to produce anthocyanins, which are used as natural food colorants and antioxidants.
• Visual Marker:
• Pigmentation can serve as a visual marker for specific developmental stages or the production of certain compounds within the callus.
• This is useful for research purposes.
• Research Tool:
• The study of pigmented callus can provide insights into the biosynthesis of plant pigments and other secondary metabolites.
• Genetic Studies:
• Pigmentation can also be used as a visual marker for genetic studies.

Examples:

• Callus cultures of certain species may exhibit red, purple, or blue coloration due to the accumulation of anthocyanins.
• Carotenoids can impart yellow, orange, or red coloration to callus tissue.
• Green callus is often observed when it originates from leaf tissue.

• May appear red, brown, purple due to pigment or stress
• Could indicate phenolic compound accumulation or senescence

Callus Characteristics to Observe

Type of Callus	Characteristics
Friable Callus	Soft, crumbly, loosely arranged; good for cell suspension culture
Compact Callus	Firm, dense, tightly packed; ideal for shoot/root regeneration
Embryogenic Callus	Appears granular; capable of forming somatic embryos and regenerating plants
Non-embryogenic Callus	Undifferentiated; lacks potential for embryo or organ development
Organogenic Callus	Shows early signs of shoot/root initiation; capable of forming organs
Pigmented Callus	Contains pigments (green, red, brown); may indicate specific metabolic activity
Mucilaginous Callus	Sticky, jelly-like due to mucilage secretion; common in species with high polysaccharide

 

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