Plasmas meet nanoparticles—where synergies can advance the frontier of medicine

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IOP PUBLISHING JOURNAL OF PHYSICS D: APPLIED PHYSICS

J. Phys. D: Appl. Phys. 44 (2011) 174018 (14pp) doi:10.1088/0022-3727/44/17/174018

Plasmas meet nanoparticles—wheresynergies can advance the frontier ofmedicineM G Kong1, M Keidar2 and K Ostrikov3,4

1 Department of Electronic and Electrical Engineering, Loughborough University,Leicestershire LE11 3TU, UK2 Department of Mechanical and Aerospace Engineering, George Washington University,Washington, DC, USA3 Plasma Nanoscience Centre Australia (PNCA), CSIRO Materials Science and Engineering,PO Box 218, Lindfield, New South Wales 2070, Australia4 School of Physics, The University of Sydney, Sydney, New South Wales 2006, Australia

E-mail: m.g.kong@lboro.ac.uk

Received 25 October 2010, in final form 6 December 2010Published 14 April 2011Online at stacks.iop.org/JPhysD/44/174018

AbstractNanoparticles and low-temperature plasmas have been developed, independently and oftenalong different routes, to tackle the same set of challenges in biomedicine. There are intriguingsimilarities and contrasts in their interactions with cells and living tissues, and these arereflected directly in the characteristics and scope of their intended therapeutic solutions, inparticular their chemical reactivity, selectivity against pathogens and cancer cells, safety tohealthy cells and tissues and targeted delivery to diseased tissues. Time has come to ask theinevitable question of possible plasma–nanoparticle synergy and the related benefits to thedevelopment of effective, selective and safe therapies for modern medicine. This perspectivepaper offers a detailed review of the strengths and weakenesses of nanomedicine and plasmamedicine as a stand-alone technology, and then provides a critical analysis of some of themajor opportunities enabled by synergizing nanotechnology and plasma technology. It isshown that the plasma–nanoparticle synergy is best captured through plasma nanotechnologyand its benefits for medicine are highly promising.

(Some figures in this article are in colour only in the electronic version)

1. Introduction

In the open literature, there have been countless scientificpublications, roadmap statements of relevant scientificcommunities and government white papers that highlightthe immense potentials of nanotechnology for healthcare.Targeting the same set of major challenges in modernmedicine (e.g. infectious diseases, cancers), low-temperaturegas plasmas have in more recent years been seen to attractrapidly growing interest in their biological applications. Sofar, the development of these two technologies for biomedicineappears to be largely independent from each other andeach has achieved some notable clinical successes. Thescale of their own potential for medicine and the extentof the scientific challenges they face as a stand-alone

technology are such that there appears little incentive toexplore possible overlaps, in terms of either underpinningscience or technological implementation, between these twoequally exciting technologies. Yet close examination showssome very intriguing contrasts in their characteristics whenconsidered to meet the same basic requirements for medicalapplications, for example the efficacy as a therapeutic agent,the selectivity against malignant cells (e.g. pathogens andcancer cells), the toxicity and indeed the safety to healthycells and tissues, and finally the targeted delivery towardsdiseased tissues. Nanotechnology has superior capability inpenetrating into a tissue, while its toxicity still representsan area of considerable uncertainties. On the other hand,biological effects of low-temperature plasmas stem from theirnon-equilibrium reaction chemistry with very high level of

0022-3727/11/174018+14$33.00 1 © 2011 IOP Publishing Ltd Printed in the UK & the USA

J. Phys. D: Appl. Phys. 44 (2011) 174018 M G Kong et al

chemical dissociation. Their interaction with a living tissue isnormally topical with limited penetration, but their cytotoxiceffects are better understood through their similarity to freeradical biology. Such fundamental contrasts invite the questionof the scale of the opportunities brought about by plasma–nanoparticle synergy and call for a detailed analysis of itsbenefits to the ultimate goal of developing effective, selectiveand safe therapies for modern medicine.

This perspective paper offers the first critical analysisof whether there is a need for a synergistic combinationof nanotechnology and plasma technology, what benefitsthe plasma–nanoparticle synergy may bring about, and howsuch synergy may be realized in practice. This analysisis built on a concise review of plasma science (section 2)and nanoparticle–cell interactions (section 3) as well as acomprehensive review of plasma–cell interactions (section 4).A number of plasma–nanoparticle synergies are suggested andanalysed in section 5, and the conclusions thus reached are usedto highlight in section 6 the landscape of major opportunitiesto advance the frontier of therapeutic medicine by capitalizingon the synergy between plasmas and nanostructures. It isemphasized that the full potential of synergizing plasmas andnanostructures can only be realized when nanoparticles arefabricated within a low-temperature plasma and delivered bythe plasma to a disease target—a technology known as plasmananotechnology.

2. Plasma science and nanotechnology

Low-temperature plasmas represent a unique thermally non-equilibrium environment where a variety of reactive species aswell as different forms of energy (e.g. thermal, electromagneticand chemical) can be produced. This ability has been widelyused for a large number of applications in nanotechnology.The most common applications are in micro- and nano-scale synthesis and processing of advanced nanomaterials.Representative examples include etching and conformalcoating of high-aspect-ratio features in a silicon waferfor nano-electronics, low-temperature synthesis and post-processing of nanopatterns and arrays of nanostructures forsensing, photovoltaics and optoelectronics, highly selectivefunctionalization and other post-processing of localizedsurface areas with nano-scale dimensions, as well as theproduction of nanoparticles with tailored features such as size,shape, facet expression and surface reactivity [1–5].

The examples of the nano-scale objects producedor post-processed using low-temperature plasmas span allthree dimensions and range from zero-dimensional Si-basedquantum dots arranged in two- or three-dimensional patternsin a host matrix and arrays of one-dimensional single-walled carbon nanotubes with controlled chirality and metaloxide nano-architectures to two-dimensional free-standing andcatalyst-supported graphene sheets and flakes and carbonnanowalls as well as self-organized patterns of shape-tunedthree-dimensional semiconducting nanostructures [6–17].The sizes and other properties of the nano-scale objects can beprecisely tailored to meet the requirements of any particularapplication.

These properties can be very different from the propertiesof nanoparticles and other nanomaterials produced usingother techniques such as thermal chemical vapour deposition(CVD) or wet chemistry. For example, the unique abilityof reactive plasmas to dissociate molecular hydrogen hasbeen successfully used to synthesize free-standing silicon andgermanium nanoparticles with nearly perfect cubic shape,which is rarely achievable otherwise [18]. Furthermore,carbon nanotubes and other one-dimensional nanostructuresshow pronounced vertical alignment which is not common tothermal CVD [19, 20].

On the other hand, effective control of any particularfeatures of the nanostructure or nanoparticles (NPs) has beenintimately related to the ability of the plasma environmentto concentrate the building units of the nano-scale matterand the associated energy in designated microscopic andnano-scale surface areas [21]. A balance of plasma-specificforces (e.g. ion drag, electrostatic, thermophoretic) has alsobeen reported as an effective tool for the precise delivery ofnanoparticles to the specified microscopic areas on the surface;this approach was also instrumental in almost completeremoval of nanoparticles away from the surface areas wheretheir deposition is not warranted [22, 23]. For many otherunique features and advantages of the plasma-based nano-scale synthesis and processing the reader should be referredto previous publications as well as to other papers in thisspecial issue.

In the following we will discuss how the above uniquecapabilities of low-temperature plasma-based nanotechnologycan be used in applications in biology and healthcare.

3. Interaction of nanoparticles with cells

Let us now consider some of the most common effectsof nanoparticles on living cells and their applications innanomedicine and nano-biotechnology. The interactionsof plasma-produced reactive species and electromagneticradiation with living cells will be considered in the next section.

Rapid development of nanotechnology over the lastdecade made it possible to synthesize different types ofnanoparticles whose diameter is of the order of a fewnanometres and even less. The surfaces of such nanoparticlescan be modified by bioactive molecules or imaging probesthat can be adsorbed, coated, conjugated or linked tothem. As such, these nanoparticles were proposed for celllabelling and targeting, tissue engineering, drug delivery, drugtargeting, magnetic resonance imaging, etc [24–28]. Wide-range applications stimulated intensive study of interactionof nanoparticles with a living tissue and in particular thepenetration and migration of the nanoparticles inside thetissue. Some authors suggested that the processes governingthe penetration of reactive radicals (see extensive discussionof these effects in section 4) [29] and those governingnanoparticles are not the same [30], making a good casefor penetration selectivity argument. In addition, penetrationefficacy of nanoparticles inside various tissues is distinctlydifferent. For instance, it was shown that nanoparticles areable to penetrate the hair follicle and stratum corneum (SC),

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but viable epidermis (VE) is reached only occasionally. Itwas concluded that those nanoparticles are unable to permeatethe skin. Titanium dioxide nanoparticles of different sizes(from 20 to 100 nm) do not penetrate to SC layers, the humanepidermis and dermis, but are solely deposited on the outermostsurface of the SC [31].

Meanwhile, metallic (e.g. gold) or metal oxide(e.g. iron oxide) nanoparticles have recently demonstrateda reasonable therapeutic efficacy, selectivity, tumour affinityand concomitant in vivo tolerance in cancer therapy [32–37].The treatments are based on targeted delivery of thefunctionalized (e.g. using glucoproteins) nanoparticles to thetumour-affected areas and using either a brachytherapeuticor hyperthermal/photothermal treatment. In the former case,low-dose electron emission from β-emitting gold-198 (198Au)nanoparticles is used while the latter case relies on localizedsurface plasmon excitation which generates significant yetlocalized heat. The nanoparticles are usually deliveredvia direct intratumoral injections and penetrate malignantcells through tumour vasculature and pores. Application ofnanoparticle treatment makes it possible to significantly reducethe size of the tumour before surgical resection and is animportant milestone towards the ultimate ability of completeand irreversible tumour resolution without surgery. This abilityhas been demonstrated in animal trials and is currently at thestage of human clinical trials [35, 36].

In parallel and out of health concerns, an additionalresearch activity stimulated by the rapid developments ofnanotechnology is study of human body contamination andhealth effects of nanomaterials (nanotoxicity) [38–42]. Thisrapidly expanding research field is commonly referred to asnano-safety.

As an example of a combination of plasma treatmentconsidered in section 4 with the nanoparticle treatment brieflydiscussed above, let us consider a simple combination ofsuitably functionalized nanoparticles and a cold plasma jet[43, 44]. In such a case, the cold atmospheric plasma (CAP)jet can have two primary purposes. Firstly, the jet willcarry nanoparticles and thus will provide their delivery tothe desired position and good localization of the treated zonegoverned by the jet cross-section. Since the intensity of theplasma jet can be limited to below the damaging thresholdto living tissues, thus the plasma jet will serve just as thedelivering agent. Secondly, it is possible to electricallycharge nanoparticles by means of fundamental mechanismof charging of bodies introduced to plasma (due to muchhigher mobility of electrons in comparison with ions). Thevalue of the charge accumulated on the nanoparticle maybe regulated by adjusting the plasma parameters and bythe location of the nanoparticle injection point (since thetimescale of particle charging in plasma and its flight timethrough the jet are comparable, both about microseconds [44]).Charging of nanoparticles in plasma enables utilization ofelectrical field for microscopic level control of nanoparticlecharacteristics. Symbioses of cold plasmas and nanoparticlescan lead to some extremely interesting results. Onevery promising example of such synergy is cancer therapy.Using antibody-conjugated nanoparticles with cold plasmas

led to the five-fold enhancement of melanoma cell deathcompared with the anticancer efficacy of the plasma treatmentalone [45]. The specific mechanisms leading to such aremarkable improvement in the anticancer efficacy are not fullyunderstood.

This paper focuses on direct uses of nanoparticlesand nanostructures in the treatment of diseases (i.e. usedas active drugs and therapy) so as to explore theirsynergy with low-temperature plasmas whose biomedicalapplications are developed primarily as a novel therapy(see section 4). It is worth noting that most biomedicalapplications of nanoparticles are related to drug deliveryand therapeutic uses of nanoparticles represent only about2% of nanomedicine publications in 1984–2004 and 3% ofnanomedicine patent filings worldwide in 1993–2003 [46].So far, 24 nanotechnology-based therapeutic products havebeen approved for clinical use with total sales exceeding $5.4billon [46]. Targeted diseases include leukaemia and cancer,infectious diseases (e.g. fungal and protozoal infections;hepatitis A, B and C), immunodeficiency diseases (e.g. HIV).While cytotoxic effects of metallic materials (e.g. silver)are employed for nanotechnology-based therapies [47], thereis increasing interest in encapsulation of nano-therapeuticagents in polymers for both treatment of diseases and theirpreventative vaccination [48]. The reader is referred to recentreview papers [48, 49].

4. Interaction of plasmas with cells

For treatment of living tissues, low-temperature plasmas are atpresent used largely for therapeutic purposes although plasma-assisted imaging is conceivable. Their biological effectsare predominately through their non-equilibrium chemistry,highly unique and otherwise difficult to access because ofthe very high degree of chemical (in particular, electron-impact) dissociation in the plasma. Low-temperature non-equilibrium gas plasmas present a unique environment ofreactive oxygen species (ROS) and reactive nitrogen species(RNS), charged particles, photons as well as heat, pressuregradients and electrostatic and electromagnetic fields, manyof which are known to induce biological effects. For example,nitric oxides (NO) are known to promote cell proliferation [50]whereas hydroxyl radicals (OH·) are very effective againstbacteria [51]. The interest in the use of non-equilibrium gasplasmas in healthcare has grown very strongly over the past10 years [52, 53], largely owing to an important technologicalbreakthrough that enables the generation of low-temperatureatmospheric pressure plasmas with well controlled thermalstability even in electronegative gases such as oxygen, airand water vapour [54–57]. Such low-temperature plasmas arecommonly known as CAPs and have been shown to be capableof effectively inactivating bacteria, fungi and virus, inducingapoptosis in cancer cells, and stimulating proliferation ofmammalian cells, all in a dose-dependent fashion [52, 53].Today, CAPs are increasingly used in an impressive arrayof healthcare applications such as blood coagulation [58],skin disinfection [58, 59] and wound disinfection [60], woundhealing [61], sterilization of surgical instruments and medical

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Table 1. Main considerations of CAP design.

Global considerations:

• CAP scalability versus localized treatment• Spatially uniform/controlled treatment• Temporally reproducible treatment• Control of gas temperature• Effect of UV, ozone and NOx

• Presence of liquid

Reaction chemistry:

• Use of charged particles versus neutral species• Role of ROS and RNS and their lifetimes• ROS/RNS transport through liquid and their generation in

liquid

Plasma–cell interactions:

• Plasma effects on cellular components• Plasma penetration through cells and tissues• Effect of electric and electromagnetic fields• Safety and toxicity• Selectivity between pathogens and cells

devices [62], cancer therapy [63] and food decontamination[64–66]. Their potential for healthcare is profound andtheir clinical successes so far are exceptionally encouraging[67, 68], thus fuelling a rapid expansion of research activitiesin an emerging but now highly visible field known as plasmamedicine. For details of the opportunity, challenge andperspectives of plasma medicine, the reader is referred toseveral recent reviews [52, 53].

4.1. Modes of plasma interaction with biological samples

With a mean-free path-length as short as a few tens ofnanometres in atmospheric pressure plasmas [69], theirtemporal stability is difficult to maintain over a large gapdistance between two electrodes and as such their electrodegap is typically a few millimetres. When a CAP is broughtclose to a sample to be treated (e.g. an infected human tissue),the sample may become a part of the plasma-containing electriccircuit. Here the discharge current flows through the sampleand charged particles are likely to play an important role. Thismode of plasma treatment is sometimes known as the directmode of plasma–sample interactions [70]. By contrast, theCAP may be used as an afterglow to treat a sample, which iselectrically disconnected from the plasma-containing circuitand experiences only little discharge current. The role ofcharged particles is much reduced and neutral reactive plasmaspecies are likely to play a more dominant role. This modeis sometimes known as the indirect mode of plasma–sampleinteractions. The choice of how a CAP may be configured tointeract with a cell-containing sample depends on many inter-dependent factors, many of which are highlighted in table 1.

Figure 1 shows some of the common CAPs currentlyused in medical applications. Here we consider the CAPjet (see figure 1(a)) [71–74] as an example and discuss howsome of the current understanding of plasma–cell interactionis used in CAP designs. Formed in a flowing gas, the CAPjet is well suited for precision and localized treatment, such

Figure 1. (a) A CAP jet treating a thumb and (b) a CAP jet arraytreating a hand, both pictures taken at Loughborough.

as root canal disinfection [75–77] and cell transfection [78].Some of the current CAP jets rely on the generation of high-intensity plasma in the upstream electrode region (sustained ina radial-directed electric field) and its axial diffusion towardsa downstream sample is often unaided. Such devices areknown as the cross-field plasma jet [79]. A combination ofan upstream localized plasma and the short lifespan of manyreactive plasma species (typically several tens of microsecondsor shorter) means that the biological efficacy of such CAPdevices is critically dependent on a very short plasma–sampledistance in the region of a few millimetres (often just 1–3 mm).Longer plasma–sample distance is desirable for downstreamintroduction of gas and liquid precursors, and for controlledtreatment of uneven surfaces. To this end, the upstreamplasma may be spatially extended by a strong axially directedelectric field [79]. With an axially directed flow of theplasma-forming gas, both charged and neutral species canbe significantly extended downstream to facilitate effectivedelivery of short-living plasma species to the sample surface.With this strategy, it is possible to extend the plasma jetlength to many centimetres and even more than 10 cm [80].The alignment of the electric and the gas flow fields in suchCAP jets (often known as the linear-field plasma jet [79])encourages better penetration of plasma species (see belowfor further discussion) into a living tissue placed downstream.Importantly, the linear-field plasma jet provides two additionalimportant advantages, when nanoparticles are driven through aplasma region for uptake of reactive plasma species (see furtherdiscussion in section 5.2). Firstly its strong axial electricfield can be used to drive the flow of the electrically chargednanoparticles. Secondly, its much longer plasma plume lengthboth enhances the uptake of reactive plasma species by thepassing nanoparticles and sustains the ROS/RNS uptake rightto the entrance point of the nanoparticles into a tissue. Thelinear-field plasma jet is therefore ideally placed for synergisticcombination with nanoparticles.

Diameter of a single CAP jet is typically a few millimetres[81]. For treatment of large samples having a width of a fewcentimetres and greater, such as chronic wounds, it is essentialto overcome the inherently small size of CAPs through scaling-up [69, 81]. This has been shown to be technologicallyfeasible, as shown in several reported up-scaled CAPssuch as the 1D and 2D CAP jet arrays (see figure 1(b))[81, 82], the microwave plasma torch [83] and the floating-electrode dielectric-barrier discharges (DBDs) [58]. While

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the development of these large CAP systems was initiallymotivated by practical considerations of scaling-up, theirinteractions with cells and tissues contained in a downstreamsample differ from each other. For example, energetic ionswith their kinetic energy reaching above 20 eV are important ina sample treated by the floating-electrode DBDs [84] whereasions are largely absent in the downstream sample treatedusing the microwave plasma torch [83]. Depending on theelectrode configurations, the CAP jet array can be made tocontrol the involvement of ions and indeed charged particles[85]. Plasma–cell interaction mechanisms are multi-facetedand at present are not fully understood. However, differentapplications are likely to be best addressed with differentup-scaled CAP sources.

It is important to note that in practice diseased livingtissues are either moist or covered by a layer of liquid (e.g. boldfluids, blood and/or wound fluids). When a CAP jet is usedto treat a living tissue, its plasma species are delivered tothe air–liquid interface and then undergo transportation, andsometimes secondary ROS/RNS generation within the liquidmedium, before reaching cells and tissues. Therefore, it isimportant to consider plasma reactions that interact with watermolecules [86–88].

4.2. Reactive plasma species and their biological targets

Reaction chemistry of a CAP device varies considerablywith chemical composition of the plasma-forming gas evenwith the same electrode configuration and the same appliedvoltage. In a study comparing an atmospheric air plasmawith its counterpart in helium/oxygen both sustained in sub-microsecond voltage pulses, it is observed that the air plasmais dominated by N∗

2 species with their emission in the UVCregion whereas the helium/oxygen plasma is dominated byoxygen atoms [89]. It is of particular interest to contrastthese against bacterial inactivation kinetics data and establisha hierarchical list of bactericidal plasma agents (and theirthreshold bactericidal doses). However, the distinct differencein reaction chemistry is found to result in a small difference ofa mere 10 s in the timescale over which the two pulsed CAPsystems need to achieve 6 log reduction of bacterial spores [90].In other words, very different combinations of plasma speciesmay yet result in similar efficacy of bacterial inactivation.Different chemical compositions of ‘lethal plasma dose’ seemto exist, each with different synergistic effects among variousplasma species, and it appears that the bactericidal effect ofa specific plasma species may be compensated by those ofothers. It is therefore highly probable that there exist manydifferent and viable recipes of bactericidal plasma chemistry,each of which may take different pathways towards bacterialdeath (e.g. protein, lipid and DNA).

In the gas phase (i.e. before reaching the cell-containingliquid medium), plasma produces UV photons and manybactericidal ROS/RNS, including singlet oxygen (O∗

2), atomicoxygen (O/O∗), superoxide (O·−

2 ) and nitric oxide (NO).In a moist or liquid environment, hydroxyl radicals (OH·)and hydrogen peroxide (H2O2) are produced. Complexphysicochemical processes in the plasma impose a limit on

Figure 2. SEM images of B. subtilis spores (a) before treatmentwith a He/O2 CAP jet and (b) after plasma treatment [100].Reprinted with permission from IEEE Publishing (© 2006 IEEE).

the maximum achievable concentration of each plasma speciesand result in a close cross-dependence between concentrationsof different plasma species. It is both intriguing and importantto note that many of these ROS/RNS are also producedendogenously within the human body itself and are involvedin its defence against bacterial infection through, for example,Fenton chemistry [91]. Therefore in a way, plasma disinfectionmimics how the body responds to bacterial infection. Havingbeen produced in plasma, ROS/RNS are then delivered tocells and tissues exogenously and their delivery is subject toatmospheric absorption of photons and recombination of short-living species outside the plasma zone. This can filter outthe effects of some ROS/RNS, since their concentrations atthe cell/tissue location may fall below the threshold of theirbiological effects. For example, current evidence of bacterialinactivation studies suggests that UV photons play a lessimportant role than ROS [92–94]. Figure 2 shows a damagedmembrane of B. subtilis spores after plasma treatment, a typicalresult of oxidation.

Upon the arrival of plasma species on the surface ofmicroorganisms, their uptake by pathogens and infected tissuesis affected by how plasma species interact with the cell-surfacestructure (e.g. receptors, membrane proteins) and whether theypenetrate into the microorganism. These factors mean that thebiological effect of each possible plasma agent is likely to bekept within a range of plasma dose and penetration depth. Thisview also applies to diseases other than bacterial infection suchas cancers.

Important insights have been gained from investigationson possible plasma-inflicted damage to key cellularconstituents. Studies using isolated protein and plasmid DNAmodels have shown protein and DNA damage [95–99], andmore relevant experiments using bacterial cells have provideddirect evidence of breaching and rupture of cell membrane[100], reduction and degradation of proteins [101–103], lipiddamage [104] and (mostly) single-strand breaks (SSBs) ofDNA [105]. These results have prompted the suggestion ofthe involvement of ROS, charged particles and UV photons[92, 100, 105–108], with ROS often linked to oxidation ofprotein and lipid and with UV photons linked to DNA damage.The view of DNA damage, particularly double-strand breaks(DSBs), being largely induced by UV and shorter-wavelength

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Figure 3. Structure of an animal cell. Reprinted from [115] by permission from National High Magnetic Field Laboratory, Florida StateUniversity, USA.

radiation has its root in radiation biology where many hundredDSBs per microorganism are not uncommon among irradiatedbacteria [109]. The prevailing view of bacterial killing inradiation biology as ‘death by DNA damage’ for the past 50years is now challenged and is being replaced by the alternativeview of ‘death by protein damage’ for which the role of ROSand RNS is becoming increasingly important [110]. Thisdevelopment in radiation biology may become important ininforming and formulating the direction and priority of futurestudies of plasma-mediated mechanisms against pathogens. Itshould have a similar impact on studies of plasma-safety forhealthy mammalian cells and those of plasma-mediated killingof cancer cells.

The long-standing attempt to understand plasma inactiva-tion mechanisms has very often suffered a common lack of anappreciation that even the same plasma source can have verydifferent plasma chemistries as it transits, often abruptly andrapidly, from one plasma mode to another [89, 111]. Similarlyunder-appreciated is the paucity that the resistance of micro-organisms to external stresses is influenced by their micro-environment [112], an obvious example being their interactionwith the surface of their supporting substrate [113]. Futurestudies are likely to include detailed characterization of bothplasma and microbial sample conditions.

Studies of plasma inactivation mechanisms have in thepast paid little attention to the effects of the liquid environmentthat contains bacteria. Energetic electrons, ions and UVphotons can react with water molecules and produce additionalROS in the liquid medium. Equally importantly, it is possiblethat plasma treatment may release oxygen, nitrogen andhydrogen species directly from the chemical structure of cellsand tissues and the latter can then be used to alter the localreaction chemistry on the surface of cells. These processes

may be referred to as the secondary production or the on-siteproduction of ROS/RNS. They introduce both an excitingopportunity for plasma medicine and a complex challengeto the study of plasma inactivation mechanisms. From thestandpoint of how ROS and RNS are used to defend againstbacterial infection, however, the key reactive species includeO∗

2, O·−2 , OH·, H2O2 and NO and their cellular targets are

largely known [114]. These can be considered as the startinggroup of priority species in mechanistic studies. Similarprioritization of key plasma species may be achieved fortreatment of cancers.

4.3. Selectivity and safety

When plasmas are used to treat diseased living tissues wherepathogens or cancer cells co-exist with healthy mammaliancells and tissues, an important challenge is to achieve plasmaselectivity with lethality against pathogens but little damageto healthy mammalian cells and tissues. This is one of themost important questions in plasma medicine but surprisinglyhas been scarcely addressed in detailed in vitro investigationsalthough histological data from ex-vivo and in vivo experimentswith animals have so far shown limited damage [52, 60].Mammalian cells have a different structure to bacteria (seefigure 3), and their responses to external stresses are also likelyto be different. In radiation biology, bacteria and pathogens aremore resistant to radiation than mammalian cells. Whether thedesired reversal exists for plasma-produced ROS/RNS remainsboth critical and intriguing. From the limited in vitro studiesreported so far, it has been known that the selectivity windowbetween bacteria and fibroblast cells is significant againstE. coli [116] but limited against Staph aureus [117]. Usingan air plasma afterglow, a significant selectivity window is

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found against bacteria spores [90], suggesting that significantselectivity is attainable even though much more work is neededto examine the presence of possible subtle long-term damage.Similarly there is considerable scope for detailed selectivitystudies for plasma-based cancer therapies.

One practical complication to selectivity is the host–cellinteraction where bacterial or cancer cells invade and infect aninitially healthy tissue and then may penetrate into the bulkof the tissue thus escaping the impact of the exogenouslyapplied plasma treatment [118]. Bacterial penetration intoand co-existence with the host tissues means that the sameexternally applied plasma dose may be received differentlyby the pathogens and their host tissues thus complicatingthe selectivity established from experiments using cell media.While this presents an uncertainty, a useful indicator of itspossible consequence is that some non-thermal atmosphericplasma jet systems have already been used in minimal invasivesurgery, for example those for removing diseased tissues[67, 68, 119, 120]. These clinical successes provide a criticalcontext of realism and relevance with which to understandthe broad nature of the long-term effects of low-temperatureatmospheric plasmas [121].

4.4. Plasma penetration

The issue of migrating bacteria into the bulk of a livingtissue raises the question of plasma penetration. Estimatingfrom the half-lives of the main plasma species, the maximumpenetration depth of plasma ROS/RNS into a liquid mediumis perhaps at most a few tens of micrometres, depending onphysiological conditions of the liquid medium. For example,OH·− radicals are known to propagate for no more than a fewangstrom in liquid [110, 122] whereas the singlet oxygen hasa half-life of only a few microseconds in liquid [123]. On theother hand, H2O2 are long-lived and can diffuse throughout thecell [110, 122] even though their reactivity is only modest and,against some pathogens, ineffective. Penetration of ROS/RNSinto the skin or a living tissue is even more limited. Therefore,the plasma treatment is characteristically topical.

The topical character of plasma treatment is an advantagefor localized surgery where spatial control of damage tohealthy tissues is critical [119, 120]. For living tissuesinfected exogenously such as open wounds, plasma treatmentof the tissue surface may be applied frequently to containsurface infection. Similarly, topical plasma treatment isuseful for melanoma tumours. When a diseased tissue islocated considerably away from the skin surface, the topicalnature of plasma treatment becomes a serious limiting factor.Greater plasma dose may be effective for infected tissues lyingimmediately below the skin or wound surface, but the issue ofplasma toxicity would become a counteracting factor. Thereis clearly a need for assisted plasma penetration.

It is known that adjustment of pH may considerablyincrease the half-lives of some ROS [108], such as O·−

2 whichcould be made to last up to 10 000 s in liquid [124]. Tosee whether such extended half-lives of plasma species couldenable some ROS to penetrate through the skin and tissues,we note that there are openings and channels in the skin to

Figure 4. Structure of skin with an open wound [126]. Reprinted bypermission from Macmillan Publishers Ltd, 2008.

allow diffusion of small molecules and other externally appliedagents such as plasma species. These include the lipidicintracellular routes (5–36 nm in diameter) and trans-follicularroutes (10–210 µm in diameter) [125]. The SC and VE ofthe skin (see figure 4 and [126]) are about 32 µm and 65 µm,respectively, and the length of an aqueous channel through theskin is much greater than the combined depth of SC and VE.We assume that the effective aqueous channel is in the orderof 1 mm and the diffusion coefficient of superoxide anionsin liquid is in the order of D = 8 × 10−5 cm2 s−1 [127].For a lifetime of 10 000 s, the diffusion length of O·−

2 is2(Dt)0.5 = 17.9 mm. In fact, the lifetime of O·−

2 would onlyneed to be more than 32 s for their passage through a 1 mm longaqueous channel. Therefore, it is possible for reactive plasmaspecies to survive their passage through aqueous channelsin living tissues and reach a deep-embedded disease siteif the physiological conditions of the aqueous environmentcould be appropriately adjusted. In addition, it is possible totransiently increase the diameter of lipidic intracellular routesusing, for example, electroporation. This should enable agreater passage efficacy of plasma ROS/RNS through aqueouschannels of living tissues. It is worth noting that the deliveryof plasma ROS through the membrane of a mammaliancell is also possible as indicated in early in vitro evidenceof cell permeation of plasma species [78]. Penetration ofplasma species through the membrane of a bacterial cell ispossibly more difficult because of much narrower channelsthrough the membrane (∼ up to a few tens of nanometres indiameter).

The use of such long-living ROS as pre-madeantimicrobial or anticancer agents implies that liquids afterplasma pre-treatment become a drug [128, 129]. In thiscontext, research of plasma medicine is becoming the study ofhow plasma-enabled drugs may be used effectively and safely(i.e. plasma pharmacy) as well as the study of mechanisms ormodes of actions with which plasma-enabled drugs cure theirintended diseases (i.e. plasma pharmacology). It is knownthat endogenous ROS and antioxidants are often unbalancedin immune-compromised patients and yet their balance iscritical in the defence of a patient against bacterial infectionand in regulation of many essential biochemical functions.

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Table 2. Properties of CAPs and nanoparticles.

CAPs Nanoparticles

Reactivity ROS/RNS, UV charged particles Intracellular ROS,metal, nanomaterials

Selectivity Potentially large Can be very high subject to appropriatefunctionalization/bioconjugation

Toxicity Possibly controllable Varied, depending on material,structure, surface chemistry, etc.

Penetration Topical Penetrating

The possibility of pre-prepared long-living plasmas speciesis exciting, since they can be used as a novel and uniqueagent to address an imbalance between endogenous ROS andantioxidants thus allowing a patient to fight more effectivelyagainst infection and cancer. In addition to directly producedplasma ROS/RNS, it is possible to produce energetic ions ata streamer head [84] and/or energetic electrons in plasmasformed in a micro-scale cavity [130, 131] deep inside a livingtissue. Such energetic charged particles may be extended intoa tissue from a parent plasma outside the tissue, and they allowfor an in-tissue production of ROS/RNS since their kineticenergy is sufficient to react to water molecules and somechemical bonds of the tissue structure. As a result, they can alsobe used to address potential imbalance between endogenousROS/RNS and antioxidants. Such in-tissue production ofROS/RNS may be considered as long-range effects of plasmaspecies, and they are almost completely uncharted as an areaof investigation but represent a very important route for plasmapenetration and indeed plasma pharmacy.

In summary, CAPs are shown to be capable ofinactivating microorganisms and cancer cells through uniquenon-equilibrium chemistry. They are exceptionally versatile,offering different modes of interacting with a cell-containingsample, and they employ various reaction chemistries tounlock different pathways towards an intended therapeuticobjective (e.g. skin disinfection, wound healing and cancertherapy). With appropriate use of plasma chemistry, selectivityappears to be attainable with controlled safety. CAPtreatment is normally topical, ideally for localized cell andtissue manipulation as demonstrated in clinical successes ofendoscopic surgery procedures. However, there are excitingopportunities to enhance or induce significant penetrationof plasma species into a living tissue, with energetic ionsand electrons potentially capable of triggering an in-tissueproduction of ROS/RNS in a disease area. In addition, plasmapre-treatment of a physiologically optimized liquid medium(e.g. using pH adjustment) may allow the in-liquid productionof ROS/RNS that last sufficiently long to survive their passagethrough tissue. The delivery of both energetic charges (forin-tissue ROS production) and pre-prepared plasma ROS(for penetrating to a remote site) offers a great opportunityto work with the chemistry of endogenous ROS/RNS, byaddressing the potential imbalance of endogenous ROS/RNSand antioxidants. The role of CAPs as a complementaryregulator of free radical biology within the body may openup a whole new host of opportunities to treat human diseases.

5. Synergies between plasma and nanoparticles

Both nanoparticle technology and cold plasma technologyoffer exceptional opportunities to biomedicine. There isgrowing momentum that drives forward each technologytowards a full realization of their immense promise tohealthcare, and there is significant evidence that eachtechnology is likely to influence very strongly how modernmedicine may be practiced in future. While the exceptionalscale of these opportunities tends to encourage a focus on onetechnology alone, there may be even greater opportunities intheir synergy. Given that CAPs have so far been used largelyfor therapeutic purposes, our discussion of synergy is confinedto the treatment of diseases.

5.1. Comparison of CAPs and nanoparticles

For treating diseases, medical applications of CAPs andnanoparticles often require at least four considerations, namelytheir reactivity, their selectivity against pathogens or cancercells, their toxicity to healthy mammalian cells and tissues,and their penetration to the contaminated regions. Table 2is a summary of the properties of CAPs and nanoparticleswhen compared against the above four considerations. CAPsprovide non-equilibrium reaction chemistry with many oftheir main reactive species also present in the body withknown effects on bacterial and mammalian cells [114]. Thebiological implication of plasma chemistry is not dissimilarto those related to free radical biology. Provided that futurestudies result in appropriate understanding and control ofplasma dosage, it is highly probable to achieve selectivekilling of pathogens or cancer cells with little damage tohealthy mammalian cells and tissues. There have been clearsuccesses of some CAP-based clinical procedures [119–121].On the other hand, nanoparticle-based therapies are yetto enjoy a comparably similar level of clinical acceptancedespite far greater extent of scientific advances [132] andthis is partly related to the current anxiety over theirtoxicity. Many nanoparticles generate ROS when incubatedwith different cells or when inoculated in vivo [133], andnanotoxicity represents both a key area of research and asignificant uncertainty [134]. Notwithstanding future progressof nanotoxicity research, it is highly desirable to reduce theminimum amount of nanoparticles necessary for a targetedtherapeutic effect. This is one of many areas where plasma–nanoparticles synergy is particularly beneficial.

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Figure 5. Fabrication of nano-capsule with enclosed plasma ROS and/or RNS, a polymer coat, and conjugated surface-borne,protein-binding molecules, fully completed in a CAP (jet) environment.

5.2. Synergy in reaction chemistry

Consider the in vitro study discussed in section 3 in whichantibody-conjugated nanoparticles were used to enhanceplasma-mediated killing of melanoma cells [45]. Itmay have benefited from a synergetic combination of theanticancer properties of both CAPs and antibody-conjugatednanoparticles. This synergy can and should be taken advantageof to reduce the minimum plasma dose against cancer cells andas a result reduce plasma toxicity. In reversal of roles, plasmascould be used as a supplementary agent to assist nanoparticle-based therapies against cancer and/or infection. It has alreadybeen shown that plasma species induce apoptosis among cancercells (in vitro device) [52] as well as suppress the growthof melanoma and lung cancers (in vivo evidence) [63]. Soplasma pre-treatment of a tumour can reduce the defence ofits cancer cells and as a result reduce the minimum inhibitionconcentration of intratumorally injected nanoparticles whenused alone. This is yet to be reported in open literature butis anticipated from the current understanding of anticancereffects of both plasmas and nanoparticles. The obvious patientbenefit of reduced risk of nanotoxicity could be potentiallyprofound for nanoparticles-based therapies, as it is likely toalter the parametric space and indeed the paradigm of nano-safety. Furthermore, the benefit extends also to other majordiseases such as skin and wound infection, infectious diseasesand wound healing.

One exciting area of plasma–nanoparticle synergy is tocombine the unique non-equilibrium plasma chemistry withthe superior penetration of nanoparticles. Such synergy canbe realized at the stage of nanoparticle synthesis or post-synthesis processing. This is illustrated in figure 5. Supposenanoparticles are produced first with an iron core, which can bemade to acquire a high affinity to oxygen atoms and hence trapoxygen atoms, for example if the core has a porous structure ofappropriate pore size, shape and surface–volume ratio [135].

The iron nanoparticles are then coated with biocompatiblesilica (with reduced toxicity and optimized wettability) andfinally bio-conjugated by attaching specific molecules thatare designed to bind with specific proteins in a targeted cell.Such nanoparticles can be delivered to a diseased area by,for example, injection via bloodstream, and by minimizingdissolution during their passage to the diseased site. Themagnetic core of the nanoparticles can then release heat andoxygen atoms to the proteins of the targeted cell to whichthe nanoparticles are attached, by means of redox reactions.The release of oxygen atoms onto a buried site of a tumourtissue or an infected tissue induces a site-specific effect againsteither cancer cells or pathogens. While fabrication of suchmultifunctional nanoparticles is a major challenge, the useof plasmas in the fabrication of the nanoparticles allows thenanoparticles to be immersed in oxygen atoms of very highconcentrations thus achieving high uptake rate. The bonding ofoxygen atoms onto the nanoparticles needs to be not too strongso as to be released readily on the target site, yet not too loose toavoid being lost on their way to the targeted site. Nanoparticlesof different surface-to-volume ratio release oxygen atoms atdifferent locations down a tumour body or an infected tissue. Ifthe diseased tissue is subject to a concomitant plasma treatmentso that pores and other aqueous channels are temporally openedby plasma (see section 5.3), the delivery of the nanoparticlescarrying oxygen atoms will be made more efficient.

Discussion in section 4.4 indicates that some ROS(e.g. O·−

2 ) may be made to last longer and could also becarried by nanoparticles as in the case of oxygen atoms,although further work is needed to substantiate its practicality.The above discussion suggests a new concept of plasma-seeded nanotechnology in which nanoparticles are preparedwith encapsulated plasma reaction chemistry by immersingtheir fabrication in a non-equilibrium plasma environmentwhere very high concentrations of ROS and RNS can beup-taken by nanoparticles. Material properties and surface

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functionalities of nanoparticles are hardly exploited fortrapping and sustaining ROS/RNS during the productionof nanoparticles and before them being coated and bio-conjugated.

The above synergy takes advantage of the superiorcapability of nanoparticles to penetrate through the skinbarrier [30], thus liberating plasma from its limitation as anintrinsically topical technique. The potential specificity ofnanoparticles to attach to diseased cells as against healthycells [136] offers selectivity, and the use of ROS or RNScarried by nanoparticles as the main agent against either cancercells or pathogens reduces the reliance on toxic materials(e.g. silver). These anticancer or disinfecting effects arerealized selectively on a nano-scale and their study may beconsidered as nano-scale plasma pharmacy. While there areclearly significant engineering and scientific challenges, thebenefits and implications of nano-scale plasma pharmacy arevery exciting.

5.3. Synergy in cell permeation and cellular manipulation

There have been some reports of plasma-mediated cellpermeation [78], and possible mechanisms may includesurface deposition of charges, electroporation by global andlocal electric field, exothermic recombination of excitedspecies and radicals, and locally released heat. Energy and/orheat can be released by either plasmas or nanoparticles,although via different routes. Once above a certain threshold,the global effects of the released energy can contribute tocell permeation. However, localized energy release is ofgreater interest. Plasma ROS/RNS may act on specific siteson the cell surface and exothermic radical recombination islikely to be surface roughness-selective [137], both capable oflocalized deformation to the cell surface and hence inductionof enhanced cell permeation. Furthermore, plasma ROSare known to degrade adhesion proteins [138] and integrin[139]. These alter the cell–substrate and cell–cell attachment,thus deforming the cell and affecting permeation of itsmembrane channels. Equally, it is possible to achieve aspatially preferential pre-deposition of nanoparticles at specificcell-surface sites (e.g. surface proteins and integrin) so thatlocalized plasma degradation and hence cell deformation areenhanced, leading to more significant cell permeation andintracellular uptake of plasma species.

Electrically assisted or mediated cell permeation is ofparticular interest, as electroporation [140] can be inducedat the level of electric field in atmospheric plasmas. Forexample, the electric field is at the order of 0.5 kV cm−1 atthe tip of a CAP jet [74, 141] and higher at that of a plasmastreamer [84]. In RF microplasmas, the sheath electric fieldis of the order of 80 kV cm−1 [130]. The temporal openingof membrane pores, usually over a microsecond timescale,allows increased intracellular uptake of plasma species and/ornanoparticles. It is worth noting that the electric chargingtime of the membrane of a mammalian cell is about a fewhundred nanoseconds [140]. When the temporal scale of theelectric field in the plasma is shorter than the electric chargingtime of the cell membrane, strong intracellular electric field

can be established and this would lead to forced intracellularpenetration of plasma species and nanoparticles. Nanosecondpulsed CAPs are known to be possible [142, 143] and can beused to set up intracellular electric field inside a mammaliancell. This is useful in treating cancer cells. Equally,nanoparticles carrying charges could be rapidly deposited onthe cell membrane over a nanosecond scale, for examplethrough a CAP jet, and this can set up a permeating electricfield into the intracellular space. Strong intracellular electricfields not only enhance the uptake of plasma species and/ornanoparticles, but also induce other important biologicaleffects such as intracellular calcium release and enhancedgene expression [144]. Therefore, the benefits of electricallyassisted cell permeation could go beyond enhanced uptake ofplasma species and/or nanoparticles.

Interestingly, plasma-borne nanoparticles can be inten-tionally used to create a clear difference in morphologicalfeatures on the surfaces of targeted and non-targeted cells.These properly labelled and functionalized nanoparticles canbe delivered and distributed around the cellular surface thusacting as externally delivered ‘etching masks’ which are com-monly used in nano-scale plasma etching to enable selectivetreatment of the unmasked areas. Similarly, the ion-focusingeffect discussed above may lead to an effective and selectiveheating of the nanoparticles rather than the open surface areas.In this case one could expect ROS/RNS radicals to interact withthe unmasked (open) areas of the surface only. On the otherhand, selective heating of nanoparticles may lead to the con-trolled heating of the cell membrane underneath the nanopar-ticles. This may in turn induce heat-enhanced inter-diffusionof radicals between the nanoparticles and the cellular mem-brane. For comparison, these effects cannot be achieved usingfocused laser beams since the typical sizes of laser beam spotsare in the micrometre range (which is comparable to the sizesof small cells) and cannot resolve sub-micrometre features onthe cellular surfaces. By tailoring the nanoparticle sizes onecan mask and treat surface areas of virtually any dimensions,which offers exciting opportunities for unprecedented increasein selective treatment at the cellular level or even the organellelevel. The many important benefits discussed above stem fromthe opportunity to induce spatially differentiating effects, ona nanometre scale, by exploiting the synergy between plasmaspecies and nanoparticles. For future reference, this may bereferred to as plasma-nano cellular manipulation.

5.4. Synergy for enhanced penetration and selectivity

The above discussion on permeation through cellular porescan be extended to permeation through aqueous channelsthrough a living tissue, although the timescale is likely to belarger because of the larger length scale of a tissue. Surfacedeposition of energy by plasmas and/or nanoparticles can bemade spatially selective, and electrically induced temporalopening of pores and channels within a tissue is equallypossible. It has recently been shown that an impingingplasma streamer incepting a tissue moves preferentially to anearby channel in the tissue and establish a strong in-channelelectric field there [84, 145]. Consistent with the discussion

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in section 5.3, the spatially extended electric field can triggeran in-tissue production of plasma ROS/RNS well away fromthe streamer–tissue inception point. This offers an interestingroute for plasma penetration into a living tissue, particularly intissue cavities and openings in a wound by putrescence or otherdisease-caused routes. Again, ROS/RNS can be delivered intoa tissue by nanoparticles encapsulated with plasma ROS/RNS(see section 5.2).

Synergistic combination of plasmas and nanoparticlescould even facilitate selective penetration. It is known thatnanoparticles may preferentially deposit near cancer cellsrather than healthy cells [136]. Therefore, a combineduse of plasmas and nanoparticles is likely to enhance thedirectionality of the propagation of both plasma species andnanoparticles towards diseased sites within a tissue. Thereare many different implementation routes, some of whichare realized in plasma-based nanotechnology, such as mutualorientation of ion fluxes and nanoarrays [146]. This orientationis very different for different surface morphologies. If thesurface features some micro/nanostructures with reasonablylarge aspect ratios (e.g. >3–5), the effects of incoming ionfluxes and the resulting feature electric field enhancementeffects may be very different. For example, the outerlayers of many eukaryotic cells feature hierarchical brush-like structures. Different elements of this structure wouldthus draw different ion fluxes and would then be chargeddifferently. More importantly, normal and cancerous cellshave very different membrane external structure, so-calledbrush [136]. Therefore, the direct effect of the electric field(electric field enhancement) and charging is expected to bedifferent for the normal and cancerous cells thereby providingan excellent opportunity to achieve the as yet elusive selectivetreatment of normal and cancerous cells by plasma ion species.

It is possible to facilitate the charging and dischargingof different locations in a tissue surface, using plasmas,nanoparticles or their combination, in order to establishpossible ion-focusing channels for enhanced penetration ofplasma species or nanoparticles. Many of the above-discussedenhancements of plasma and/or nanoparticle penetration areenabled by charged particles and their electric field, which canbe used to charge nanometre and micrometre scale structuresand as such enable plasma chemistry on nano- and micro-features of a diseased area (either cells or tissue sites).The importance of charges and micro-scale tissue chargingprovides a unifying point for plasmas and nanoparticles—nanoparticles are most conveniently charged in plasma andmuch less useful using other means. In addition, it is possibleto control the charge polarity of nanoparticles by tuning plasmaconditions [147] and, thus, to enrich the therapeutic potentialof nanoparticles.

The enhanced penetration by synergizing plasmas andnanoparticles offers spatial selectivity and indeed a way toenhance their safe use as a therapeutic strategy. It offers anunexpected aid for up-scaling, since the selective move ofa plasma streamer to a nearby channel means that a CAPjet array may preferentially establish strong electric fieldsin many natural or putrefied channels regardless of theirrelative locations to the jet–tissue inception points. Plasma jet-specific feedback control helps maintain similar plasma–tissue

interaction [81], and the design of the CAP jet array couldbecome reasonably independent of the specific properties ofthe diseased tissue that it would be used to treat.

As one final example of synergistic approach for applica-tions other than therapies, hydroxyapatite[HA, Ca10(PO4)6(OH)2] has been widely used to promotebiological functions of various biomedical and dentalimplant/filling materials [148]. This bioceramic materialis the main mineral constituent of bone and tooth tissues,and HA coatings have revealed inspiring clinical advantagesin promoting efficient implant fixation and implant-to-boneadhesion shortly after the implantation, as well as faster boneremodelling due to enhanced bidirectional growth and for-mation of a bonding interlayer between bone and implant[149–151]. Recently, nanoparticle HA dental enamel paste hasbeen used for rapid repair of early caries lesions without theneed of removing of healthy tooth material, commonly prac-ticed in dentistry to ensure the filling sticks [152]. However,the issues of appropriate sterilization of the damaged tooth ar-eas still remain. This is where CAPs and HA nanoparticles canbe used simultaneously to implement both effects—the CAPsto be used not only as the carrier of the HA nanoparticles tothe targeted tooth but also as simultaneous sterilization of thetooth surfaces. The HA nanoparticles, precisely delivered tothe targeted growth sites, will then be used for dental tissueremodelling. This exciting synergistic opportunity awaits itsrealization.

6. Concluding remarks

Against a backdrop of truly immense progress in the currentlyparallel fields of nanotechnology and cold atmospheric plasmatechnology for biomedicine [46–49, 52–53], this perspectivepaper was motivated primarily by the intriguing contrastbetween these two well-established technologies in terms oftheir reactivity, selectivity, toxicity and targeted penetration todiseased tissues. Inevitably the latter invites the question oftheir possible synergy and the related benefits to the ultimategoal of developing effective, selective and safe therapies formodern medicine.

In terms of their underpinning science, nanomedicineis more advanced than plasma medicine. To this end,the paper started with a brief review of nanoparticle–cellinteractions and a comprehensive review of plasma–cellinteractions. A key advange of cold atmospheric plasmasis their non-equilibrium reaction chemsitry with very highlevels of chemical dissociation that is otherwise difficult toaccess and that strikes an intriguing similarity to the chemistryof endogenous ROS/RNS produced by cells themselves.Evidence of key plasma ROS/RNS and their cellular targetswas presented, and, when appropriate, linked to thoseknown in free radical biology and radiation biology. Whilenormally used as a topical treatment, cold atmospheric plasmatechnology has already found important clinical success inendoscopic surgery with good indication of safety for healthycells and tissues. For treatment of a diseased area buried insidea living tissue, the technological viability of several plasmapenetration techniques was suggested and assessed. These

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included the in-tissue production of ROS/RNS by micro-scaleplasmas penetrating into aqueous channels in a tissue whilstreacting with water molecules and chemicals bound to thetissue structure. Another technique was direct penetrationof physiologically adjusted liquids with their antimicrobialor anticancer effects induced by plasma pre-treatment. Suchplasma-treated liquids thus form a novel form of drugs, andtheir studies were referred to as plasma pharmacy and plasmapharmacology, an important avenue of future investigationsof biomedical applications of plasmas. Plasma-mediated cellpermeation was also discussed.

When plasmas and nanoparticles are combined, a numberof important benefits were shown to emerge, such as reducedtoxicity through combinatorial antimicrobial or anticancereffects of plasmas and nanoparticles. Given the current focuson safe use of nanoparticles, this synergy was indicated asparticularly beneficial in reducing the minimum inhibitionconcentration of nanoparticles for cancer cells or pathogens.To advance one step further, one exciting opportunity ofsynergy was shown to be related to the fact that porousnanostructures may be used to trap reactive plasma species(e.g. oxygen atoms) before the nanostructure is encapsulatedwith a bio-polymer coat and then bio-conjugated. Coupledwith the possibility that some plasma ROS may be made toextend their half-lives and so survive their passage to a diseasedtissue, nanoparticles could become a carrier of pre-preparedplasma ROS/RNS. This was shown to be a powerful strategy,partly because plasma ROS/RNS could be delivered deep intothe diseased tissue or even into an infected mammalian cell(thus overcoming the topical character of plasma treatment)and partly because less chemically reactive materials may beused for manufacturing nanoparticles thus mitigating the issueof nanotoxicity. While there remain considerable fabricationchallenges, one implementation route was thought to involvenanoparticles being prepared in an atmospheric plasma jetwhere nanopaticles are immersed in very high concentrationsof plasma ROS/RNS for efficient uptake and are chargedsimultaneously for subsequent directional penetration intotissues towards cancer or infected cells. These suggest thepossibility of nano-scale plasma pharmacy.

Upon the arrival on the region of malignant cells, it wasshown possible for nanoparticles to target specific cellularsites, for example, by difference in sub-microscale features onthe cell surface and by mutual orientation of plasma ion fluxesand nanoarrays. Also shown possible was the pre-depositionof nanoparticles onto specific surface sites of cell membraneto form a spatially selective cell-surface mask for spatiallyselective treatment of subsequently applied plasmas. Theseopportunities could lead to an unprecedented selectivity ofplasma/nanoparticle treatment at the cellular level or even theorganelle level. This suggests a highly desirable capabilityof nano-scale plasma manipulation of cell, and it wouldmost likely materialize when the plasma technology and thenanotechnogy are synergistically combined. As an illustrationof the power of plasma–nanoparticle synergy beyond directtreatment of major diseases, the use of nanoparticles forenhancing bone and tissue remodelling was considered. Thebenefits of this important nanotechnology would be enhanced

significantly if a simultaneous step of plasma sterilizationwas used.

In essence, the synergy of low-temperature plasmas andnanoparticles aims to take advantage of distinct and differentstrengths of each of the two technologies in terms of reactivity,selectivity, safety and disease targetting. Its benefits go wellbeyond simple combinations of the two technologies andindeed can only be fully exploited when nanoparticles aresynthesized and then post-processed/functionalized within theplasma environment, because of the need for in situ accessto high density plasma ROS/RNS and because of the benefitsof charged nanoparticles for preferential cell targeting. Theplasma–nanoparticle synergy not only enables a step changein their performance matrix for biomedical applications, butalso unlocks unexpected doors to many currently unknownopportunities. These combine to offer an unprecedented grandopportunity with potentially enormous impact on modernmedicine.

Acknowledgment

MGK acknowledges partial support from the EnglishDepartment of Health and the Engineering and PhysicalSciences Research Council (UK). MK was supported in partby NSF/DOE Partnership in Plasma Science and Technology(NSF grant CBET-0853777, DOE grant DE-SC0001169).KO acknowledges partial support of CSIRO’s OCE ScienceLeadership Scheme and the Australian Research Council.

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