Challenges facing sterilization and depyrogenation of nanoparticles: Effects on structural stability and biomedical applications Melissa A. Vetten Msc, Clarence S. Yah PhD, Tanusha Singh PhD, Mary Gulumian PhD PII: DOI: Reference:
S1549-9634(14)00135-X doi: 10.1016/j.nano.2014.03.017 NANO 925
To appear in:
Nanomedicine: Nanotechnology, Biology, and Medicine
Received date: Revised date: Accepted date:
11 December 2013 1 March 2014 29 March 2014
Please cite this article as: Vetten Melissa A., Yah Clarence S., Singh Tanusha, Gulumian Mary, Challenges facing sterilization and depyrogenation of nanoparticles: Effects on structural stability and biomedical applications, Nanomedicine: Nanotechnology, Biology, and Medicine (2014), doi: 10.1016/j.nano.2014.03.017
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ACCEPTED MANUSCRIPT Challenges facing sterilization and depyrogenation of nanoparticles: Effects on structural stability and biomedical applications 3,4
, Tanusha Singh (PhD)5,6, Mary Gulumian
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Melissa A Vetten (Msc)1,2, Clarence S. Yah (PhD)
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(PhD)1,2 *
Biochemistry and Toxicology Section, National Institute for Occupational Health, National
Department of Molecular Medicine and Haematology, School of Pathology, University of
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Health Laboratory Services, Johannesburg 2000, South Africa
the Witwatersrand, Johannesburg 2000, South Africa 3
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Human Sciences Research Council, Newton Park 6055, South Africa Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health,
Immunology & Microbiology Section, National Institute for Occupational Health, National
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Baltimore MD 21205, USA
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Health Laboratory Services, Johannesburg 2000, South Africa Department of Immunology, School of Pathology, University of the Witwatersrand,
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Johannesburg 2000, South Africa Corresponding author: Melissa Vetten P O Box 4788 Johannesburg 2000; Tel: +27 (11) 712-6428; Fax: +27 (11) 712-6429 E-mail:
[email protected] Word count for abstract: 108 Word count for complete manuscript: 4890 Number of references: 34 Number of figures/tables: 4 tables 1
ACCEPTED MANUSCRIPT The authors acknowledge the financial support from the Department of Science and Technology, South Africa and the National Institute for Occupational Health.
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The authors declare that there are no competing interests.
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ACCEPTED MANUSCRIPT Challenges facing sterilization and depyrogenation of nanoparticles: effects on structural stability and biomedical applications
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Abstract
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This review outlines and compares techniques that are currently available for the sterilization of nanoparticles and addresses the topic of endotoxin contamination. Several techniques are available for
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the removal of microbial contamination from nanoparticles developed for use in nanomedicine applications. These techniques include filtration, autoclaving and irradiation, as well as formaldehyde, Of these sterilization methodologies, filtration may
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ethylene oxide and gas plasma treatments.
potentially remove microbial contamination without altering the physicochemical properties of the carrier nanoparticles, nor affecting their toxicity and functionality. However, no single process may be applied to all nanoparticle preparations and, therefore, it is recommended that each nanoparticle-drug
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system be validated on a case-by-case basis.
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Introduction
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Key words: nanoparticles, sterilization, endotoxin
The unique physical and chemical properties of nanoparticles (NPs) have resulted in substantial
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research being performed on their potential applications in various fields including biology and medicine. Nanomedicine applications include the development of “lab-on-chip” technology in order to assess biomarkers, drug and gene delivery, tissue engineering, and cancer therapy (1-2). Any nanoparticle-drug based formulation requires the solvent to be sterile and apyrogenic, in addition to being safe, non-toxic and non-irritating for both in vitro and in vivo applications. The term sterility refers to the absence of viable microorganisms that could pose a risk when administered. The current accepted sterility assurance level (SAL) is limited to10-6, that is to say not more than one viable microorganism in one million parts of final product is allowed (3). Manufacturers of medical devices are required to ensure that their products meet established quality requirements and specifications, including regulations regarding microbial contamination. Potential sources of microbial 3
ACCEPTED MANUSCRIPT contamination during production of pharmaceutics include the raw materials, equipment and processes used during production, in addition to the facility and personnel (4).
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Potential contaminating microorganisms include bacteria, fungi and mould; however the removal of endotoxins, the lipopolysaccharides in the cell membrane of Gram-negative bacteria, must also be addressed. Magalhães and colleagues (5) provided a review of endotoxins and their removal from
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biological preparations. They discussed the pathophysiological effects of endotoxins, as caused by
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activation of the immune system and release of pro-inflammatory mediators, which lead to endotoxin shock, tissue injury and sometimes death. Since the effects of endotoxin are related to the amount of endotoxin present, in the case of drug products, this would imply that the endotoxin limit would be dependent on the amount of drug product administered to the patient. The formula for the endotoxin
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limit provided by the Food and Drug Administration (FDA) is K/M; where K is 5.0 EU/kilogram, and M is the maximum recommended dose of product per kilogram of body weight administered in one
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hour (6).
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Although numerous well-established sterilization techniques exist, concerns have been rasied regarding the adverse effects that these techniques may have on the physicochemical characteristics of
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the nanoparticles. A change in these characteristics could potentially affect both the toxicity and the efficacy of the sterilized nanoparticles. This paper reviews various sterilization methodologies that have been assessed for the removal of microbial and endotoxin contamination from nanoparticle preparations with the aim of providing a possible recommendation on a suitable methodology for nanomedicine production and sterilization.
Methodologies implemented for the sterilization of nanoparticles A literature survey of publications obtained from reputable journals shows that several conventionally used methodologies such as filtration, autoclaving, irradiation, as well as treatment with
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ACCEPTED MANUSCRIPT formaldehyde, ethylene oxide and gas plasma, have been implemented for the sterilization of
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nanoparticles.
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Filtration
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Sterile filtration is a commonly used method for the physical removal of microorganisms from chemically and thermally sensitive liquids, through the use of 0.22 µm membrane filters. This
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technique has been shown to be widely applicable as it does not appear to have any adverse effects on the nanoparticles (Table 1). Poly(-caprolactone) (PEC) nanospheres of mean diameter below 200 nm were successfully sterilized by filtration with 0.2 μm cellulose acetate membrane filters without alteration of their size, morphology or concentration (7). Filter sterilization through 0.22 µm filters
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has also successfully been implemented for the sterilization of PEGylated poly(γ-benzyl-L-glutamate)
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(PBLG) NPs (8) and polyester NPs (9).
The use of 0.2 µm or 0.22 µm filters may not always be possible if the NPs are larger than, or close
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to, the pore size of the filters since clogging can occur resulting in a decreased yield (10-11). For
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example, filtration of 200-300 nm poly(DL-lactide-co-glycolide) (PLGA) nanospheres prepared using the standard Emulsion Solvent Diffusion (ESD) method resulted in less than 10% of the nanospheres passing through the membrane filter (12). This problem was circumvented by optimising the synthesis methodology to produce nanospheres with a particle diameter of 103-163 nm, of which 100-98% could pass through the membrane filter and successfully pass bacterial sterility tests. However adjusting the size of the nanoparticles in order to enable them to pass through a membrane filter may not always be feasible. Sterile filtration may therefore present itself as a reasonable method for the removal of bacterial contamination, provided that a sufficiently high percentage of nanoparticles can be recovered following filtration. 5
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Autoclaving Autoclaving kills microbes with high pressurized steam, at a minimum temperature of 121 °C, within
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15–20 minutes depending on the size of the load and the contents to be sterilized. This methodology has been shown to have a number of effects on the nanoparticles sterilized (Table 2). Fesharaki and colleagues synthesised selenium nanoparticles using Klebsiella pneumonia bacteria followed by
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recovery of the nanoparticles from the bacteria by autoclaving at 121 oC, 17 psi for 20 min. Energy-
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dispersive spectroscopy (EDS) confirmed chemical stability of the selenium nanoparticles before and after sterilization (13). On the other hand, autoclaving PBLG NPs at 121 °C for 20 min resulted in the aggregation and a drastic increase in size of these nanoparticles (8). Further investigation of the effect of autoclaving on aggregation was conducted on PEC nanospheres where it was shown to be
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dependent on the polymer or surfactant used during synthesis. Nanospheres prepared with Cremophor RH40 showed aggregation following autoclaving, whilst those prepared with Pluronic remained the
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same size (7). The manner of synthesis was also shown to influence effects of autoclaving on hydroxyapatite (Hap) nanoparticles, where nanoparticles synthesised using wet chemical synthesis
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(WCS) showed significant aggregation and morphology change following autoclaving, in comparison to the Hap nanoparticles produced by hydrothermal synthesis (HS) which showed no change (14). In
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addition, the surface charge of the HS Hap nanoparticles became less negative due to autoclaving, whilst the charge of the WCS Hap nanoparticles remained unchanged.
The effects of various sterilization techniques on the physicochemical properties of poly(ethylene glycol) (PEG) coated gold nanoparticles (PEG-AuNPs) and tiopronin coated gold nanoparticles (tiopronin-AuNPs) was determined by transmission electron microscopy (TEM), ultraviolet-visible (UV-Vis) spectroscopy, and Fourier transform infrared (FTIR) (15). Autoclaving of the PEG-AuNPs produced minor aggregation of particles and no changes in the PEG coating. In contrast, autoclaving of tiopronin-AuNPs resulted in the growth of the particles from approximately 2 nm to 5 nm, a process likely caused by a growth and recrystallization process known as Ostwald ripening. A 6
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increase
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particle
size
was
also
observed
after
autoclaving
unloaded
polybutylcyanoacrylate NPs (16).
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Amphiphilic β-cyclodextrin (β-CDC6) nanospheres and nanocapsules, in addition to their Tamoxifen citrate loaded counterparts, aggregated and increased in size, possibly due to heat-induced chemical modifications of β-CDC6 (10). This disruption of NP integrity resulted in a significant decrease in the
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quantity of Tamoxifen citrate entrapped by both the nanospheres and the nanocapsules. On the other
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hand, it has been reported that sterilization at 121°C of either drug-free or drug-loaded solid lipid nanoparticles (SLNs) had no significant effect on NPs (17).
Apart from physicochemical changes that may or may not arise during autoclaving, this method may
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also produce changes in the biological effects of the sterilized nanoparticles that should be investigated. Autoclaving of citrate-stabilized silver nanoparticles at 121°C for 30 min has been
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shown to have no significant effect on the size or morphology of the NPs, as determined by dynamic light scattering (DLS) and TEM. Despite this, the autoclaved NPs showed an increased tendency to
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cause platelet aggregation, a sensitive in vitro indicator of thrombogenicity (18). Dry and wet autoclaving of titanium dioxide (TiO2) nanotubes was also shown to differently affect the adhesion,
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proliferation and alkaline phosphatase activity of osteoblasts (19). Finally, in vitro cytotoxicity studies on Cyclosporine A-loaded SLNs, which were autoclaved at 110°C for 30 min, showed no toxicity, whereas nanoparticles autoclaved at 121°C for 15 min demonstrated significant toxicity. This increase in toxicity was explained to be due to release of the surfactant Tween 80 during sterilization at higher temperatures, which lead to cell damage (20).
The majority of literature reviewed on sterilization via autoclaving showed that the aggregation of nanoparticles, as well as changes in their biological activities, is of concern. Moreover, it would appear that these effects are not only dependent on the type of nanoparticles and their coating, but also on the methodology and reagents used during their synthesis. 7
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Irradiation Sterilization using irradiation can be achieved through gamma (γ) irradiation, electron beam (e-beam),
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X-rays, UV light irradiation or through the use of subatomic particles. The most common form used due to its high penetration is γ rays, which are generated by the self disintegration of Cobalt-60 (60Co) or Cesium-137 (137Cs). The advantages of this technique include its application for the sterilization of
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many materials since it is independent from chemicals or heat, and it leaves no residue after
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sterilization. E-beam sterilization is also commonly used where very high electron energy is used for a shorter duration than γ-irradiation. UV light irradiation is generally only used for sterilization of surfaces using a germicidal lamp (21). γ-rays and e-beams provide very efficient disinfection since they are able to penetrate through materials but both can cause damage to the material being sterilized
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(22). An irradiation dose of ≥15 kGy of either γ-irradiation or e-beam irradiation has been shown to be effective in the sterilization of poly(butyl cyanoacrylate) (PBCA) NPs that were deliberately
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contaminated with spore-forming bacteria (23).
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Despite the efficacy of this mode of sterilization to kill bacteria, various adverse effects have been observed on nanoparticles when irradiation was used for their sterilization (Table 3). Where both γ-
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and e-beam irradiation at 10, 15, 25, and 35 kGy were used to sterilize doxorubicin-loaded or empty shell PBCA NPs, no effect on size or molecular mass could be observed. Only the drug-loaded NPs sterilized by γ-irradiation at 35 kGy could show an increase in the molecular mass of PBCA and the polydispersity (23). In another study, a decrease in pH and an increase in molecular weight of PEC nanospheres could be observed, with no alteration in particle size when sterilized by γ-irradiation. Moreover, this sterilization method also caused the degradation of both the antimicrobial agent thimerosal and the anti-sedimentation agent hydroxyethyl-cellulose which had been added to the nanosphere formulation (7). A study of citrate-stabilized silver NPs demonstrated that γ-irradiation at 15, 25 and 50 kGy had a significant effect on the size and morphology of the NPs. Following γirradiation, NPs were shown to lose their typical polycrystalline structure and form both smaller 8
ACCEPTED MANUSCRIPT particulates and larger irregular aggregates (18). In addition, these γ-irradiated NPs exhibited a fourto five-fold increase in their ability to cause platelet aggregation and, therefore, γ-irradiation would
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not be suitable in the sterilization of these nanoparticles.
On the other hand, no significant changes in size were reported following γ-irradiation of either blank or Tamoxifen citrate loaded β-CDC6 nanospheres and nanocapsules. Although slight changes in zeta
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potential were observed, this did not result in any significant changes in nanoparticle properties. In
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addition, it had no effect on the amount of drug entrapped by the NPs or on the in vitro drug release profile, thereby demonstrating that γ-irradiation could be applied for the sterilization of these NPs (10). Other studies have also been conducted on the sterilization of microparticles developed as drug delivery systems. For example, a study investigating the effects of β- and γ-irradiation on gentamycin-
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loaded collagen/PLGA microparticle composites showed a decrease in molecular weight and glass transition temperature following sterilization. However, these changes did not influence the
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morphology or the gentamycin release profile from the composites (24). Similarly, a decrease in molecular weight of PLGA microspheres was observed after γ-irradiation without any changes in
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tetracycline-HCl content (25). Despite the changes observed following sterilisation in these examples, irradiation could still potentially be implemented for sterilization since the changes observed did not
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appear to adversely affect the attached drug, thereby maintaining the end-use function of the drug delivery system.
γ-irradiation could, however, be used for the sterilization of biosensors where, although γ-irradiation was shown to degrade polysilicon wire pH sensors covered with 3-aminopropyltriethoxysilane/silica NP nanocomposites, the authors found that the sensors could be restored following γ-irradiation using UV annealing (22). One study has used UV irradiation at room temperature for 12 h to sterilize PEGand tiopronin-AuNPs. Although effective at preventing bacterial growth, this sterilization approach could only be applied to PEG-AuNPs as the properties remained unaltered. The tiopronin-AuNPs displayed varying degrees of aggregation and coalescence of irregular shapes (15). 9
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The studies described above demonstrate that irradiation is possibly an option for sterilization of drugloaded nanoparticles. However, irradiation does often appear to have adverse effects on the NPs and it
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would therefore be necessary to individually validate each NP-drug system separately.
Formaldehyde Treatment
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Formaldehyde is an organic solution commonly used as a disinfectant and as a fixative. There is
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limited information concerning its use for sterilization of NPs for subsequent use in biomedical applications (Table 4). Formaldehyde sterilization is useful for materials that are sensitive to high temperature; however its major limitation arises from its toxicity and carcinogenicity (21). Formaldehyde treatment of lyophilized PBCA NP powders at 60°C showed increases in size and
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clumping following resuspension of the NPs (16). Formaldehyde treatment at 60°C for 1 hr showed slight aggregation of PEG-AuNPs, whilst tiopronin-AuNPs showed significant aggregation and the
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formation of larger, irregularly shaped particles (15). Exposure of the formaldehyde-sterilized tiopronin-AuNPs to human cell line U937 in vitro resulted in a significant decrease in cell viability.
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This was attributed either to the observed aggregation and changes in the shape of the nanoparticles, or to formaldehyde residues present following sterilization. Due to both these adverse effects,
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formaldehyde may not be recommended for sterilization of nanoparticles.
Ethylene Oxide Treatment Ethylene oxide (EO) treatment is widely used for the sterilization of medical devices. EO acts as a strong alkylating agent which denatures nucleic acids and functional proteins of microorganisms, thereby presenting itself as an excellent sterilization methodology (26). In terms of its disadvantages, EO is flammable and explosive, and EO and its residues are also toxic and carcinogenic (21, 26).
Not many studies have been conducted to order to investigate the effect of EO sterilization on nanoparticles (Table 4). It has been reported to be a good methodology for the decontamination of 10
ACCEPTED MANUSCRIPT copper/low-density polyethylene (Cu/LDPE) nanocomposite T-shaped intrauterine devices (IUDs), which contain 15.0 wt.% copper nanoparticles. The sterilization was performed at 50±5°C, with a relative humidity of 60%, and an EO concentration of 500 mg/L, for an exposure time period of 2 hr.
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This treatment did not affect the internal structure, the surface functional groups, the mechanical properties, or the release of cupric ions from the device when tested 14 days after sterilization (27).
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Similarly, PEG- and tiopronin-AuNPs sterilized with EO at 54°C for 60 min showed no alteration in
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the structure or size distribution, assessed by TEM and UV-Vis spectroscopy, or in the coating material determined by the vibration peaks of FTIR (15). However, EO sterilization of gentamycinloaded collagen/PLGA microparticle composites not only resulted in aggregation and changes in the gentamycin release profiles, but also produced chemical changes in the gentamycin (24). It is possible
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that the same mechanism that makes EO so highly efficient for sterilization may introduce changes in NPs and their associated drugs. The addition of alkyl groups during sterilization may render the
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microorganisms nonviable (26), however it may also introduce chemical and structural changes to
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NP-drug systems, thus removing EO as an ideal mode of sterilization of nanoparticles.
Gas Plasma Method
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Plasma can be defined as ionized gas that has properties of both gases and liquids, and has been shown to have a broad spectrum of antimicrobial effect. Although the mechanism of action of gas plasma sterilization still needs further research, it is believed that it is related to oxidation and reduction effects on microbial structures (28). Advantages of this methodology include its use at low temperatures and its non-toxicity (29). Effects on nanoparticles were observed in one study using this methodology (Table 4), where the effect of the gas plasma technique was investigated on PEG- and tiopronin-AuNPs. No structural changes of the tiopronin coating or the size and size distributions were observed. This same treatment, however, could cause aggregation, as confirmed by TEM, as well as alteration of the PEG coating, as confirmed by FTIR, possibly due to the highly oxidative nature of the gas plasma procedure (15). Gas 11
ACCEPTED MANUSCRIPT plasma sterilization may therefore not be an ideal method for sterilization of NPs because most of their bio-conjugates are prone to oxidation.
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Endotoxin contamination
Endotoxin contamination presents itself as a further challenge that must be addressed prior to administration of NPs to human subjects. Currently the in vitro limulus amoebocyte lysate (LAL) test
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is the assay of choice for the determination of endotoxin contamination. The LAL assay exists in three
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formats, namely chromogenic, turbidity, or gel-clot assessments. A study has shown that even when formal acceptance requirements for the validity for the LAL assay are met, dramatic differences in the results between two different LAL assays for the same nanoparticle can occur (30). Subsequently underestimation or overestimation of endotoxin concentration may be reported as a result of
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interference with the assay system. Nanoparticles could interfere with the reactivity of the endotoxin with the assay, with the LAL reaction itself due to the catalytic properties of the NPs, or with the
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detection of the reaction products due to NP absorbance, luminescence or fluorescence. It is therefore suggested that when results from different LAL assays differ by more than 25%, the results should be
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verified by the in vivo rabbit pyrogen test (30). Moreover, the inclusion of appropriate inhibition or enhancement controls are essential to help recognise whether negative results are due to absence of
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endotoxin, or inhibition of the assay (31).
Endotoxins will easily pass through a 0.22 µm membrane filter that relies exclusively on size exclusion as a retention mechanism. The pharmaceutical industry therefore makes use of several other techniques to remove endotoxin such as various types of chromatography and ultrafiltration, depending on the characteristics of the product to be sterilized (5). For example, polymyxin B affinity columns are very effective in removing endotoxin contamination due to their very high binding affinity for lipid A, which is the glycolipid component of lipopolysaccharide (LPS). It has however been observed that the use of polymyxin B columns to remove LPS contamination from nanoparticles may also lead to the loss of these nanoparticles, especially those that may have high surface binding 12
ACCEPTED MANUSCRIPT affinity to LPS (32). Nanoparticles have a large surface area to volume ratio which enhances their reactivity and affinity to bind contaminants on their surfaces. Smaller magnetite NPs (100 nm), due to their
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larger surface area (33).
The charge of the nanoparticles may also determine how they will interact with Polymyxin B affinity
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columns. Endotoxins are mainly negatively charged and therefore have been shown to have especially
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strong binding affinity for positively charged cyclic peptide Polymyxin B (34). Charges on the surface of nanoparticles will not only affect the binding of the endotoxin to the nanoparticle, but also alter the ability of the nanoparticle to pass through the positively charged Polymyxin B column.
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In summary, to date, no efficient technique has been established to remove endotoxin due to the complexity of LPS and nanoparticles. Among the challenges being discussed regarding the removal of
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LPS contamination from nanoparticles is the extreme heterogeneity of LPS in its composition and structure, differences in size, its thermostability, and its insensitivity to pH changes (32). The FDA
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recommendations of applying either high temperatures (250°C for 45 min) or high concentrations of acids or bases would be effective in removing LPS, but these conditions would most likely also affect
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the properties of nanoparticles. The ultimate choice would therefore be to keep the production process contaminant free in order to eliminate the problem of removing endotoxins from the nanoparticles. Such contaminant-free processes would involve the use of ultrapure water, gloves, fume hoods, and the cleaning of the work area and glassware with PyroCLEANTM prior to synthesis. By implementing these precautions, LPS contamination could be lowered by 16 - 24 fold in the production of spherical gold nanoparticles (32).
Conclusion This review has covered the sterilization methods used for various nanomaterials and the effects of the processes implemented on the properties of the NPs as well as on the attached drugs. The sterilization 13
ACCEPTED MANUSCRIPT methodologies discussed have been shown, where tested, to be effective in the removal or destruction of microbial contamination. However, they are frequently shown to alter the physicochemical properties of NPs, the stability of the attached drug, the drug-release profiles, and the toxicity of the
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sterilized NPs.
Filtration presents itself as a preferable option for sterilization, provided that sufficient NP can be
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recovered from the filtrate. Irradiation appears not to affect the drug release but seems to affect the
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physicochemical properties of the carrier NPs. Autoclaving resulted in aggregation of the NPs in most cases. The limited information available on formaldehyde, EO and gas plasma techniques suggest that they should not be used due to either their toxicity or their ability to alter the properties of the drugNP system. It is therefore of vital importance that the toxicity and functionality of the NPs following
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sterilization be assessed. From all the sterilization methodologies listed, no single process can equally
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be applied to all NPs and it is recommended that each NP-drug system to be validated individually.
Regarding the removal of endotoxins from NPs, the ideal situation would be to prevent contamination
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Disclaimer
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by maintaining a contaminant-free synthesis process.
The review reflects the views of the authors who are liable for the details and accuracy of the information herein and do not necessarily reflect the official views or policies of any institution or body. This article does not constitute standard specification nor is planned to design, construct, request for permit purposes. Any trade name used was solely for information and not for product endorsement.
Acknowledgements The authors acknowledge the financial support from the Department of Science and Technology, South Africa, and the National Institute for Occupational Health. 14
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List of Abbreviations 60
Cobalt-60
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Co
Cesium-137
AuNPs
gold nanoparticles
β-CDC6
Amphiphilic β-cyclodextrin
Cu/LDPE
Copper/low-density polyethylene
DLS
dynamic light scattering
e-beam
electron beam
EO
Ethylene oxide
EDS
Energy-dispersive spectroscopy
ESD
Emulsion Solvent Diffusion
FDA
Food and Drug Administration
FTIR
Fourier transform infrared
Hap
hydroxyapatite
HS
hydrothermal synthesis
LPS
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intrauterine devices
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IUDs LAL
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137
limulus amoebocyte lysate lipopolysaccharide
NP
nanoparticle
PBCA
poly(butyl cyanoacrylate)
PBLG
poly(γ-benzyl-L-glutamate)
PEC
poly(ɛ-caprolactone)
PEG
poly(ethylene glycol)
PEG-AuNPs
poly(ethylene glycol) coated gold nanoparticles
PLGA
poly(DL-lactide-co-glycolide)
SAL
sterility assurance level 15
solid lipid nanoparticles
TEM
transmission electron microscopy
TiO2
titanium dioxide
tiopronin-AuNPs
tiopronin coated gold nanoparticles
UV-Vis
ultraviolet-visible
WCS
wet chemical synthesis
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Oh S, Brammer KS, Moon K-S, Bae J-M, Jin S. Influence of sterilization methods on cell
RI P
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behavior and functionality of osteoblasts cultured on TiO 2 nanotubes. Materials Science and Engineering: C. 2011;31(5):873-9.
Gokce EH, Sandri G, Bonferoni MC, Rossi S, Ferrari F, Guneri T, et al. Cyclosporine A
SC
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MA NU
loaded SLNs: evaluation of cellular uptake and corneal cytotoxicity. Int J Pharm. 2008 Nov 19;364(1):76-86. 21.
Sílíndír M, Özer AY. Sterilization Methods and the Comparison of E-Beam Sterilization with
Gamma Radiation Sterilization. FABAD Journal of Pharmaceutical Sciences. 2009;34:43-53. Lin JJ, Hsu PY. Gamma-ray sterilization effects in silica nanoparticles/gamma-APTES
ED
22.
nanocomposite-based pH-sensitive polysilicon wire sensors. Sensors (Basel). 2011;11(9):8769-81. Maksimenko O, Pavlov E, Toushov E, Molin A, Stukalov Y, Prudskova T, et al. Radiation
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sterilisation of doxorubicin bound to poly(butyl cyanoacrylate) nanoparticles. Int J Pharm. 2008 May
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22;356(1-2):325-32.
Friess W, Schlapp M. Sterilization of gentamicin containing collagen/PLGA microparticle
25.
AC
composites. Eur J Pharm Biopharm. 2006 Jun;63(2):176-87. Bittner B, Mader K, Kroll C, Borchert HH, Kissel T. Tetracycline-HCl-loaded poly(DL-
lactide-co-glycolide) microspheres prepared by a spray drying technique: influence of gammairradiation on radical formation and polymer degradation. J Control Release. 1999 May 1;59(1):23-32. 26.
Mendes GC, Brandao TR, Silva CL. Ethylene oxide sterilization of medical devices: a review.
Am J Infect Control. 2007 Nov;35(9):574-81. 27.
Xia X, Wang Y, Cai S, Xie C, Zhu C. Will ethylene oxide sterilization influence the
application of novel Cu/LDPE nanocomposite intrauterine devices? Contraception. 2009 Jan;79(1):65-70.
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ACCEPTED MANUSCRIPT 28.
Shintani H, Sakudo A, Burke P, McDonnell G. Gas plasma sterilization of microorganisms
and mechanisms of action. Exp Ther Med. 2010 Sep;1(5):731-8. Moisan M, Barbeau J, Crevier M-C, Pelletier J, Philip N, Saoudi B. Plasma sterilization.
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Methods and mechanisms. Pure Appl Chem. 2002;74(3):349-58.
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Dobrovolskaia MA, Neun BW, Clogston JD, Ding H, Ljubimova J, McNeil SE. Ambiguities
in applying traditional Limulus amebocyte lysate tests to quantify endotoxin in nanoparticle
Smulders S, Kaiser J-P, Zuin S, Van Landuyt KL, Golanski L, Vanoirbeek J, et al.
MA NU
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formulations. Nanomedicine (Lond). 2010 Jun;5(4):555-62.
Contamination of nanoparticles by endotoxin: evaluation of different test methods. Particle and Fibre Toxicology. 2012;9(41). 32.
Vallhov H, Qin J, Johansson SM, Ahlborg N, Muhammed MA, Scheynius A, et al. The
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importance of an endotoxin-free environment during the production of nanoparticles used in medical applications. Nano Lett. 2006 Aug;6(8):1682-6. Bromberg L, Chang EP, Alvarez-Lorenzo C, Magarinos B, Concheiro A, Hatton TA. Binding
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2010 Jun 1;26(11):8829-35.
Anspach FB. Endotoxin removal by affinity sorbents. J Biochem Biophys Methods. 2001 Oct
AC
30;49(1-3):665-81.
19
ACCEPTED MANUSCRIPT Table 1.Summary of the effects of filtration on nanoparticles Type of
Mean
Effect of sterilization
of filter
nanoparticle
approximate on nanoparticle
Authors
sterilization method
RI P
size Poly (-
130 nm; 160-
No aggregation and no
Not tested
(7)
caprolactone)
180 nm
effect on the
No change in size or
No detectable
(8)
polydispersity index.
growth of
Slight change in zeta
bacteria, yeast
potential.
or fungi
No clogging of the
No bacterial
membrane filters.
contamination
SC
0.2 µm
Outcome of
T
Pore size
nanoparticle
nanospheres
0.22 µm
PEGylated poly (γ-
50 nm
benzyl-Lglutamate)
Polyester
CE
nanoparticles
sub 200 nm
PT
0.22 µm
ED
nanoparticles
MA NU
morphology
Poly(DL-lactide-
size distribution
200-300 nm
co-glycolide) nanospheres
No significant change in particle size and
AC 0.2 µm
(9)
103-163 nm
Less than 10 % of
Passed
nanospheres passed
bacterial
through filter.
sterility test
(12)
100-98% of nanospheres passed through filter.
20
ACCEPTED MANUSCRIPT Table 2. Summary of the effects of autoclaving on nanoparticles. Mean
Effect of
and
approximate sterilization on sterilization
Duration of
size
nanoparticle
Aggregation of
RI P
Autoclaving
Authors
Poly (-caprolactone)
130 nm; 160-
min
nanospheres
180 nm; 220
method
Not tested
(7)
Massive
No
(8)
SC
121°C for 20
nanoparticles dependent on
MA NU
nm
Outcome of
T
Temperature Type of nanoparticle
polymer or surfactant. Decrease in pH
ED
observed.
121°C for 20
PEGylated poly (γ-
min
benzyl-L-glutamate)
aggregations of
detectable
nanoparticles
the
growth of
nanoparticles,
bacteria,
changes in
yeast or
polydispersity
fungi
AC
CE
PT
50 nm
index and zeta potential.
121°C for 20
Amphiphilic β-
All
min
cyclodextrin
nanoparticles
nanoparticles:
205 nm
showed
Blank nanosphere
290 nm
aggregation
Blank nanocapsule
Tamoxifen citrate
Not tested
(10)
and increased 235 nm
polydispersity
21
ACCEPTED MANUSCRIPT
310 nm
index.
Tamoxifen citrate
Significant
loaded nanocapsule
reduction of
RI P
Tamoxifen
T
loaded nanosphere
citrate
entrapment. Selenium nanoparticles
245 nm
Not tested
(13)
Not tested
(14)
No bacterial
(15)
stability
MA NU
min
Chemically
SC
121°C for 20
according to energydispersion
Hydroxyapatite
WCS:
WCS:
min
nanoparticles
needle-like
agglomeration,
synthesised by either
shape,
morphology
crystallite
change,
size 17 nm
crystallite
HS: rod-like
growth.
shape,
HS: no
crystallite
significant
size 38 nm
morphology
CE
PT
120°C for 20
ED
spectroscopy.
AC
WCS* or HS†
change, surface charge less negative.
134°C for 40
PEG-AuNPs‡
60 nm
Minor
22
ACCEPTED MANUSCRIPT min
aggregation of Tiopronin-AuNPs§
2 nm
growth
some PEG-
RI P
effect on
T
AuNPs. No
poly(ethylene
glycol) layers.
SC
Tiopronin-
MA NU
AuNPs were slightly bigger. No effect on tiopronin
ED
molecules.
Unloaded
Ranged from
min
poly(butylcyanoacrylate) 116-524 nm
increase in
nanoparticles
particle size.
CE
PT
121°C for 20
Significant
Solid lipid nanoparticles, Diameter in
Minor changes
min
with or without
water;
in size,
Diazepam
trehalose;
polydispersity
Acidan N12
Pluronic
index and zeta
nanoparticles
F38:
potential
Behenic acid
60 nm; 85
dependent on
nanoparticles
nm; 78 nm
lipid used and
Stearic acid
70 nm; 90
aqueous
nanoparticles
nm; 85 nm
solution.
55 nm; 56.5
Presence of
AC
121°C for 15
Not tested
(16)
Not tested
(17)
23
ACCEPTED MANUSCRIPT nm; 76 nm
diazepam did not influence stability.
Citrate-stabilized silver
20, 40, 60,
No significant
min
nanoparticles
and 80 nm
change in size
Not tested
(18)
Not tested
(19)
Not tested
(20)
RI P
T
121°C for 30
or morphology.
SC
Increased
MA NU
tendency to cause platelet aggregation in vitro.
Titanium dioxide
autoclaving
nanotubes
100 nm
PT
(Details not
30, 50, 70,
ED
Dry and wet
autoclaving methods result in changes in cell adherence
CE
provided)
Different
to the
AC
nanotubes, which was dependent on nanotube size
110°C for 30
Cyclosporine A loaded
min, or
solid lipid nanoparticles
225.9 nm
At 110°C, no changes in
121°C for 15
particle size or
min
polydispersity, and no toxicity observed using 24
ACCEPTED MANUSCRIPT Neutral Red assay.
RI P
increase in
T
At 121°C,
particle size and toxicity
MA NU
SC
was observed.
* WCS: wet chemical synthesis † HS: hydrothermal synthesis
AC
CE
PT
§ AuNPs: gold nanoparticles
ED
‡ PEG-AuNPs: poly(ethylene glycol) coated gold nanoparticles
25
ACCEPTED MANUSCRIPT Table 3. Summary of the effects of irradiation on nanoparticles
irradiation
Mean
Effect of
Outcome
Author
approxima
sterilization on
of
s
te size
nanoparticle
sterilizatio
T
Type of nanoparticle
RI P
Type of
n method
Poly(-caprolactone)
130 nm;
Decrease in pH. Not tested
(2.5 MRad)
nanospheres
160-180
Increase in
(7)
SC
γ-irradiation
AC
CE
PT
ED
MA NU
nm; 220 nm poly(ɛcaprolactone) molecular weight, however no change in nanoparticle diameter. Thimerosalcontaining formulations showed degradation. Hydroxyethylcellulose containing formulations showed depolymerisati on. 26
ACCEPTED MANUSCRIPT γ-irradiation
Amphiphilic β-cyclodextrin
All
at room
nanoparticles:
nanoparticles
temperature
Blank nanosphere
205 nm
showed:
to deliver a
Blank nanocapsule
290 nm
No effect on
dose of 1.88
Tamoxifen citrate loaded
T
RI P
size or 235 nm
nanosphere
polydispersity. Slight changes
Tamoxifen citrate loaded 310 nm
UV
PT
ED
MA NU
nanocapsule
PEG-AuNPs*
CE
irradiation at
temperature for
Tiopronin-AuNPs†
in zeta
potential. No significant change in drug entrapment or drug release profile.
60 nm
No effect on
No
PEG-AuNP
bacterial
2 nm
(15)
growth Aggregation of
AC
room
(10)
SC
kGy/h
Not tested
tiopronin-
approximate
AuNPs.
ly 12 hr γ-irradiation
Citrate-stabilized silver
20, 40, 60
Dramatic
at room
nanoparticles
and 80 nm
changes in the
temperature
size,
(20-23oC) to
morphology
deliver 15,
and
25, or 50
biocompatibilit
Not tested
(18)
27
ACCEPTED MANUSCRIPT kGy.
y of the nanoparticles.
γ-irradiation
Polysilicon wire pH sensors
Silica
Degradation
of a total
coated with 3-
particle
from γ
dose 25
aminopropyltriethoxysilane/si
size: 14 nm
irradiation
kGy,
lica nanoparticle
followed by
nanocomposite
(22)
No changes in
Sterilizatio
(23)
size of either
n of
particle. No
bacteria
changes in
achieved
molecular mass
with a
with doses up
dose ≥ 15
doses of 10,
to 25 kGy.
kGy
15, 25, 35
Irradiation at
irrespectiv
35 kGy lead to
e of type
increase in
of
molecular mass
irradiation.
MA NU
restored by UV
annealing at room temperature Empty poly(butyl
and e-beam
cyanoacrylate) nanoparticles
PT
ED
γ-irradiation
301 nm
irradiation at Doxorubicin-loaded
poly(butyl cyanoacrylate)
temperature
nanoparticles
AC
CE
ambient
kGy.
RI P
SC
could be
UV
to deliver
T
Not tested
245 nm
annealing.
and polydispersity of drug-loaded nanoparticles. 28
ACCEPTED MANUSCRIPT
T
* PEG-AuNPs: poly(ethylene glycol) coated gold nanoparticles
AC
CE
PT
ED
MA NU
SC
RI P
†AuNPs: gold nanoparticles
29
ACCEPTED MANUSCRIPT Table 4: Summary of the effects of sterilization on nanoparticles treated with formaldehyde, ethylene oxide, and gas plasma.
method
Mean
Effect of
Outcome of
Author
approximate
sterilization
sterilizatio
s
size
on
T
Type of nanoparticle
RI P
Sterilization
n method
Formaldehyd
PEG-AuNPs*
60 nm
60° for 60
Tiopronin-AuNPs†
2 nm
PT
ED
min
MA NU
e treatment at
SC
nanoparticle Minor
No bacterial
aggregation
growth
of some PEGAuNPs. Aggregation of tioproninAuNPs.
Unloaded
Ranged from
Powders
e treatment at
poly(butylcyanoacrylate
116-524 nm
showed
60°C
) nanoparticles
impaired
(lyophilized powder)
resuspension
CE
Formaldehyd
AC
(15)
Not tested
(16)
No effect on
No bacterial
(15)
either
growth
characteristic s and increase in size.
Ethylene
PEG-AuNPs
60 nm
oxide treatment at
Tiopronin-AuNPs
2 nm
Tiopronin-
54°C for 60
AuNPs or
min
PEG-AuNPs 30
ACCEPTED MANUSCRIPT morphologies or coatings.
Copper/low-density
Not specified.
No influence
ethylene
polyethylene
Nanocomposit
on the
oxide
nanocomposite
e intrauterine
internal
treatment at
devices
structure,
50±5°C, 60%
consisted of
humidity, for
45°C for 50 min
PT
CE
treatment at
PEG-AuNPs
Tiopronin-AuNPs
AC
Gas plasma
PEG-AuNP
No bacterial
(15)
aggregation.
growth
RI P
surface
15.0 wt%
functional
copper
groups,
nanoparticles.
mechanical
ED
2 hr
(27)
SC
MA NU
relative
Not tested
T
500 mg/L
property and cupric ion release rate of the devices.
60 nm
2 nm
Alterations of poly(ethylene glycol) layer. No effect on TioproninAuNPs
* PEG-AuNPs: poly(ethylene glycol) coated gold nanoparticles †AuNPs: gold nanoparticles
31
ACCEPTED MANUSCRIPT Graphical Abstract This review summarizes the techniques available for the sterilization of nanoparticles and outlines
T
their potential drawbacks. In addition, the matter of endotoxin contamination and the need for sterile
AC
CE
PT
ED
MA NU
SC
methodology can be applied across all nanoparticle types.
RI P
synthesis is addressed. Sterilization methods need to be validated on a case-by-case basis as no single
32
