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Plasma synthesis of carbon-based nanocarriers for linker-free immobilization of bioactive cargo Miguel Santos, Praveesuda Michael, Elysse Filipe, Alex Chan, Juichien Hung, Richard Tan, Bob Lee, Minh Huynh, Clare Hawkins, Anna Waterhouse, Marcela M.M. Bilek, and Steven Garry Wise ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.7b00086 • Publication Date (Web): 05 Jan 2018 Downloaded from http://pubs.acs.org on January 9, 2018

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ACS Applied Nano Materials

Plasma synthesis of carbon-based nanocarriers for linker-free immobilization of bioactive cargo

Miguel Santos a,b,c*,‡, Praveesuda L. Michael a,b, ‡, Elysse C. Filipe a,b, Alex H.P. Chan a,b, Juichien Hung a,b, Richard P. Tan a,b, Bob S.L. Lee a,b, Minh Huynh f, Clare Hawkins b,g,h, Anna Waterhouse b,e,i, Marcela M. M. Bilek c,d,e,* and Steven G. Wise a,b,* a

Applied Materials Group, The Heart Research Institute, Newtown, NSW 2042, Australia

b

Sydney Medical School, University of Sydney, NSW 2006, Australia

c

Applied & Plasma Physics Group, School of Physics, University of Sydney, NSW 2006, Australia

d

School of Aerospace, Mechanical and Mechatronic Engineering, University of Sydney, NSW 2006, Australia e

Australian Institute of Nanoscale Science and Technology, University of Sydney, NSW, 2006, Australia f g

Australian Centre for Microscopy & Microanalysis, University of Sydney, NSW 2006, Australia Inflammation Group, The Heart Research Institute, Newtown, NSW 2042, Australia

h

Panum Institute, Department of Biomedical Sciences, University of Copenhagen, 2200 København, Denmark, i

Cardiovascular Medical Devices Group, The Heart Research Institute, Newtown, NSW 2042, Australia

KEYWORDS dusty plasmas, plasma polymerization, carbon, functionalization, multifunctional nanoparticles, nanotechnology, theranostics

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ABSTRACT

Multifunctional nanoparticles are increasingly employed to improve biological efficiency in medical imaging, diagnostics and treatment applications. However, even the most well-established nanoparticle platforms rely on multiple step wet-chemistry approaches for functionalization often with linkers, substantially increasing complexity and cost, while limiting efficacy. Plasma dust nanoparticles are ubiquitous in space, commonly observed in reactive plasmas and long regarded as detrimental to many manufacturing processes. As the bulk of research to date has sought to eliminate plasma nanoparticles, their potential in theranostics has been overlooked. Here we show that carbon-activated plasma polymerized nanoparticles (nanoP3) can be synthesized in dusty plasmas with tailored properties, in a process that is compatible with scale up to high throughput, low-cost commercial production. We demonstrate that nanoP3 have a long active shelf life, containing a reservoir of long-lived radicals embedded during their synthesis that facilitate attachment of molecules upon contact with the nanoparticle surface. Following synthesis, nanoP3 are transferred to the bench, where simple one-step incubation in aqueous solution, without the need for intermediate chemical linkers or purification steps, immobilizes multiple cargo that retain biological activity. Bare nanoP3 readily enter multiple cell types and do not inhibit cell proliferation. Following functionalization with multiple fluorescently labeled cargo, nanoP 3 retain their ability to cross the cell membrane. This paper shows the unanticipated potential of carbonaceous plasma dust for theranostics, facilitating simultaneous imaging and cargo delivery on an easily customizable, functionalizable, cost effective, and scalable nanoparticle platform.


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INTRODUCTION

Over the last decade, the number of nanoparticle platforms proposed for customized nanomedicine has vastly increased

1-2

. Nanocarriers have been designed to address a great number of clinical

conditions using cocktails of therapeutic drugs, nucleic acids, photosensitive dyes and diagnostic agents

2-5

. These platforms aim to control the biodistribution, enhance the efficacy, or otherwise

reduce toxicity of biologics or drugs by improving solubility, half-life, or delivery 2. Clinically available nanoparticle systems, including polymeric, liposomal, and nanocrystal formulations, have largely failed to meet expectations for efficacy improvements 6. Other commonly used platforms in development including gold, silver, iron oxide, silica and carbon-based nanoparticles share issues of cytotoxicity (limiting dose concentrations), sub-optimal cell penetration, and the requirement for complex chemical functionalization schemes to attach cargo. As such, there is a trend towards the development of more complex, multi-functional materials, adding increased surface functionality to improve homing, imaging or deliver drugs. With current platforms, this adds significant complexity and cost, greatly limiting the feasibility of this approach 7.

For a successful translation into the clinic, multifunctional nanoparticles should be easy to functionalize

3

and made cost-effectively 7. However, commonly proposed nanocarriers utilize

materials that are hydrophobic, bio-inactive and insoluble and thereby do not allow a direct and robust conjugation with biomolecular cargo. Functionalization strategies with chemical crosslinkers have been widely exploited to impart surface functionalities on various nanoparticle materials such as gold, metal-oxides, ceramics, polymer nanoparticles and semiconductor quantum dots

8-11

. Alternative conjugation strategies involve the pre-conjugation of molecules with the


3


nanoparticle material during self-assembly

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. Further approaches utilize mesoporous polymeric

materials with large surface to volume ratios, which electrostatically entrap high loads of molecular cargo

13

that can be released in targeted cells upon photo-chemical stimulation

14

. However,

common to all wet-chemistry approaches is the requirement for time-consuming, expensive, multistep protocols to achieve a robust conjugation between the nanocarrier surface and the molecular cargo.

Moreover, optimization, reproducibility and control over the concentration and thickness of surface coatings with functional linkers is difficult to achieve. Typically, the terminal groups of the linkers also limit the range of biomolecules that can be immobilized 9. Such functionalization approaches rely on solvents and multiple purification steps that compromise the native conformation and functionality of the molecular cargos as well as presenting safety and disposal difficulties. The use of solvents during functionalization also requires a phase transfer to aqueous solution for biomedical applications, adding additional processing steps, such as ligand exchange and modification to stabilize and disperse the nanoparticles in solution 8. Despite the recent rapid growth of nanomedicine research, the need for scalable nanofabrication strategies that can deliver novel products with improved performance, functionality and safety remains. Ideally, the rational design of nanoparticles should strive for multiple functionalization using simple approaches 3 such as direct incubation with biomolecule solutions. Amongst carbon-based nanoparticles for instance, nanodiamonds

15

and carbon quantum dots

16

are preferred in theranostics due to their low

cytotoxicity and imaging capabilities. However, while the scalable synthesis of these forms of carbon remains a challenge, post-synthesis purification can represent up to 40% of all production


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costs

15

. Moreover, off-the-shelf carbon-based nanoparticles also require extensive surface

modification with stabilizers and functional groups to achieve binding of molecular cargo.

To address the limitations of current approaches, we have developed a novel methodology based on plasma polymerization (PP) in carbonaceous dusty plasmas. Dusty plasmas are suspensions of solid particles immersed in ionized gases

17

. Plasma dust particles can be formed spontaneously

through reactive plasma polymerization of particular precursors such as acetylene methane

20

18

, silane 19 or

. The formation of charged plasma dust particulates strongly affects the fundamental

processes governing the discharge manufacturing

22

21

and presents a source of contamination in plasma-assisted

and fusion devices

23

. Dusty plasmas are ubiquitous in space

24-25

and

carbonaceous nanoparticles produced in acetylene dusty plasmas have been proposed as analogues to the dust grains formed in the interstellar medium 26. While plasma dust particles have been well studied and their dynamics in the plasma characterized

21, 27-28

, the properties enabling them to

directly immobilize multiple functional cargo for applications in theranostics have been overlooked.

Here we propose for the first time that dusty plasmas can be tailored to produce multifunctional plasma polymerized nanoparticles (nanoP3) with unique functionalization capabilities. We show that plasma polymerization can be operated in a suitable parameter window to synthesize nanoP 3 in a controlled fashion. By identifying relevant nanoparticle growth mechanisms, we further demonstrate that significant features of nanoP 3, such as yield, size, aggregation, fluorescence, surface topography, chemistry and charge can be tailored and reproducibly attained. We found that nanoP3 retain many of the favorable surface characteristics that we previously reported for


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hydrophilic and biocompatible thin film coatings 29. Longed-lived radicals and functional moieties are formed and preserved within the nanoP3 structure, enabling a simple one-step surface immobilization of bioactive cargo with wide ranges of molecular weights and chemical structures. We demonstrate the binding ability of nanoP3 in aqueous solutions by simply incubating biomolecules with wide ranging of molecular weights, structures and biological functions such as drugs, fluorescent dyes, enzymes, ribonucleic acids and antibodies. We further show that nanoP 3 is readily up-taken by multiple cell lines without the need for permeabilization and with negligible effect on cell proliferation and morphology.

RESULTS AND DISCUSSION nanoP3 synthesis and growth mechanisms We synthesized and collected nanoP3 in a radiofrequency (rf) plasma polymerization reactor, previously developed for the deposition of biofunctionalizable plasma-activated coatings 29. Three distinct phases, illustrated in Figure 1, were identified during the synthesis of nanoP3: formation and nucleation of primary carbonaceous nanoclusters (PCN) by radical/ion chain plasma polymerization (phase 1), rapid cluster aggregation (phase 2) and nanoP 3 removal and collection from the active plasma (phase 3). The synthesis process was monitored using non-invasive optical emission spectroscopy (OES), which recorded the plasma emission fingerprint during each run. We monitored the rotational-vibrational band-heads (Figure 2a) associated with electronic deexcitation of molecular species including excited N2 neutral molecules, N2+ molecular ions and CN radical molecules. These molecular species resulted from the excitation, ionization and


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dissociation of the gaseous mixture in the reactor, comprised of C2H2 (monomer), N2 and Ar, which were identified as critical in the formation of nanoP 3.

During phase 1, the activation of the initial gaseous mixture is driven by collisions with energetic electrons that oscillate in the rf electric field. This activation forms an array of different reactive species, such as ions and radicals, that combine and nucleate to form PCN with a size of up to ~10 nm

18, 20, 30

. The continuous formation and build-up of PCN in the plasma bulk was observed in

phase 1 by an increase in the overall plasma emission intensity over time (Figure 2b). As the PCN reach a critical density in the plasma 28, they rapidly aggregate into spherical-like nanoP3 (phase 2). During this second phase, we found that the aggregation of PCN coincided with a rapid increase in the N2 and N2+ emission intensities (Figures 2b). The overall increase in the emission intensity is mostly driven by a modification of the ionization profile in the plasma and by scattering of radiation by nanoP3. Changes to the ionization profile are prompted by a reduction of the electron density, necessary to compensate for the rapid increase in the electron temperature as electrons sink into the growing particles

28

. While the overall plasma emission intensity is expected to

increase during cluster aggregation 21, we found that the spectral band-head associated with CN radicals decreased in intensity during phase 2 (Figure 2b black line and black arrow). The decrease in the CN population overlaid with a 5-fold increase in the collected amount of nanoP 3 (Figure 2b open squares), suggests that the formation of nanoP3 is further assisted by accretion of radical molecules into the growing particles.

Since the dynamics of nanoP3 is governed by their mass and charge, the balance of forces acting on the particles can be modulated by the input discharge parameters, enabling optimization of


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particle dynamics and collection yields. Under our reactor conditions, the electrostatic force, which confines the particles in the plasma, can be overcome by the ion drag force with a substantial increase in the ionization degree. The rapid increase in ion density during phase 2 (Figure 2b red line and red arrow) yields a 3-fold enhancement of the ion drag force, dragging nanoP 3 out of the active plasma as they reach a critical radius (> 25 – 50 nm) and mass (~10-15 g / particle) during phase 3. The falling particles were attracted to a pulsed biased electrode positioned 10 cm from the rf electrode and optimally harvested in sterile collector vials (Figure 1 and methods). The removal of nanoP3 from the plasma was observed by a decrease in the overall discharge emission. All spectral emission band-heads returned to typical particle-free baseline values registered at the beginning of each growth cycle (Figure 2b). The use of OES to monitor the synthesis of nanoparticles in plasma discharges therefore provides a simple, non-intrusive and in situ quality control diagnostic, which would otherwise be challenging to achieve in wet chemistry approaches.

While oscillations in the light emitted by the plasma have been previously reported in acetylenecontaining dusty plasmas

18, 31

, our results in Figure 2c show that the synthesis of nanoP3 is also

oscillatory and a cyclic, continuous process with collection yields proportional to the number of cycles. We tailored the properties of nanoP3 and optimized collection yields by generating plasmas at different pressure, gas flow rate and coupled rf power. The duration of nanoP 3 formation cycles (i.e. period of the oscillations) was significantly modulated by the discharge pressure and coupled power as shown in Figure 3a. We observed an overall decreasing trend with increasing pressure and power. The formation of nanoP3 was found to occur in wider pressure windows with increasing coupled power, for example, in the range 80 – 180 mTorr at 50 W and 80 – 250 mTorr at 100W. At low pressure, the mean-free path between particles increases, reducing the collision frequency


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and the probability for the formation of PCN via radical and / or ion chain reactions. Additionally, it was necessary to increase the pumping efficiency to lower the pressure in the reactor and maintain constant total gas flow rate throughout the experiments. Higher pumping efficiency reduces the residence time in the active plasma, which also results in a lower degree of dissociation of the initial gaseous mixture. Conversely, the lack of oscillations observed at higher pressures for 50 and 75 W is most likely caused by a decrease in the energy per unit mass of the precursor, due to an excessive increase in the number density of gas molecules. Increasing the power to 100 W was necessary to extend the range at which the formation of nanoP 3 occurs, due to an increase in the energy per monomer unit mass. Results also show that the period of the oscillations decreased asymptotically with increasing acetylene flow rate within the range 0.5 – 8 sccm from 256 s to 26 s (Figure 3b) and was highly modulated by the coupled power at lower flow rates. Moreover, differences in the period of oscillations are less significant with an increase of the flow rate due to a decrease in the overall energy per monomer unit mas and residence time, showing that a further excess in monomer does not contribute to a higher nanoparticle production rate.

We optimized the coupled power and flow rates that provided the best monomer fragmentation conditions and maximized the yield rate of nanoP3. We observed that nanoP3 yield per unit area of the collector (Figure 3c) reached 3.2 g/hr m2 and 7.7 g/hr m2 for an acetylene flow rate of 3 sccm and rf coupled power of 50 W and 75 W respectively. The yield was strikingly amplified to 14.4 g/hr m2 at 100W and 6 sccm. Results demonstrate that dusty plasmas could provide a scalable nanoparticle synthesis route compatible with commercial applications, as current commercially available functional nanoparticles are typically available for research and development applications in 1 – 10 mg vials.


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Physical properties of nanoP3 We next studied the morphological features of nanoP 3 using high resolution SEM, TEM and electron tomography (Figure 4). To reveal the whole structure of the particles, we acquired images at various tilting angles of the TEM stage as shown in Figure 4a and Movie S1 (the sample stage was tilted between -66˚ and +67˚). Image alignment and reconstruction resulted in an electron tomogram (Figure 4b) comprising a total of 900 high-resolution images of a single ≈200 nm nanoP3 particle, resulting in a “z-stack” image resolution of 0.29 nm. The electron tomogram (Figure 4b and Movie S2) and simulated volume rendering (Figure 4c), are consistent with HR-SEM (Figure 4d) and HR-TEM observations (Figure 4e), showing that nanoP3 feature a highly compacted, solid and amorphous structure (Figure 4f). The rapid aggregation of PCN during phase 2, and their subsequent (radical-mediated) binding, fuses the PCN clusters into an assembled spherical-like structure, featuring a rough cauliflower-like surface morphology. The diffusion of plasma activated radical species towards the growing particles during the aggregation phase further fuses the PCN, rendering a compacted nanoP3 structure.

The average size of nanoP3 measured by SEM was 86 ± 12 nm, 134 ± 21 nm and 199 ± 22 nm at the maximum yield rate for 100 W, 75 W and 50 W respectively (Figure 5a). The hydrodynamic size of nanoP3 increased once resuspended in solution, due to the formation of an ionic double layer, (Figure 5b and 5c) to 135 ± 27 nm, 179 ± 29 nm and 240 ± 39 nm at the highest nanoparticle yields and for 100 W, 75 W and 50 W respectively. Size characterization was then investigated in aqueous solutions prepared at various pH as shown in Figure 5d. The size of all nanoP3 populations remain virtually constant within the pH range of 2 – 7, showing that the particles are stable in


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acidic and neutral media. Particle aggregation was observed in the 8 ≤ pH < 9.5 range as demonstrated by a significant increase in the hydrodynamic size, suggesting that nanoP3 are poorly stabilized in mildly alkaline media. The size of nanoP 3 synthesized at 50 W and 75 W decreased again for pH ≥ 9.5, demonstrating that stabilization can be reversed in sufficiently high alkaline media. The polydispersity index is consistent with the hydrodynamic size measurements and followed the same trend with the solution pH (Figure 5e). Importantly, all samples were characterized by a low polydispersity index and qualified as monodisperse at physiological pH, measuring 0.06, 0.11 and 0.13 respectively.

Most nanoparticle suspensions are stable electrostatically in solution when the |𝜁| ≥ 30 mV. At isoelectric point, i.e. when 𝜁 = 0 mV, the stability of a nanoparticle suspension is typically compromised due to the formation of aggregates. The stability of nanoP 3 observed at low, intermediate and very high solution pH is driven by electrostatic repulsion between charged particles. The zeta potential of nanoP3 was measured to confirm this hypothesis and found that it was readily modulated by adjusting the solution’s pH. It ranged from 57.8 ± 3 mV, 57.3 ± 5 mV and 54.8 ± 3 mV at pH 2 to -48.0 ± 1 mV, -23.0 ± 1 mV and -3.7 ± 1 mV at pH 10 (Figure 5f) for nanoP3 formulations synthesized with a coupled power of 100 W, 75 W and 50 W respectively. All nanoP3 formulations were positively charged at neutral pH, with zeta potential higher than 30 mV, demonstrating their stability and feasibility for storage in water. Laser dynamic scattering and nanoparticle tracking analysis confirmed that all nanoparticles were stable and didn’t form aggregates when dispersed in solution (Movie S3). The zeta potential measurements are consistent with the results obtained for size and PDI. The dispersion of nanoP3 in solution at pH < 8 is stabilized by electrostatic repulsion between positively charged particles. The buildup of a positive


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charge around each nanoparticle is most likely driven by the protonation of surface functional groups, such as amines, present on the nanoP3 surface. For 8 ≤ pH < 9.5 these surface groups become progressively more deprotonated and the particles aggregate as they reach their isoelectric point. Increasing the solution to highly alkaline media, i.e. pH ≥ 9.5, renders negatively charged nanoP3 as deprotonated surface carboxylic acid groups dominate, stabilizing the nanoparticles due to the repulsion between now negatively charged particles. As shown in Figure 5f, the isoelectric point (i.e. 𝜁𝑉 = 0 mV) of nanoP3 changes for various formulations, occurring at 8 < pH < 9, 9.5 < pH < 10 and around 10 for nanoparticles produced at 50 W (3sccm), 75 W (6 sccm) and 100 W (6sccm) respectively. Since the aggregation and instability of nanoP3 is modulated by the solution pH at which the particles reach their isoelectric point (i.e. neutral surface charge), the surface chemistry of nanoP3 can be engineered such that the nanoparticles have sufficiently high net charge to repel each other at a particular solution pH.

Chemical properties and radical kinetics To study the chemical composition of nanoP 3 we carried out X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). Typical XPS and FTIR survey spectra of nanoP3 are shown in Figure 6a and 6b. Since the two main broad bands (3680 cm-1 – 2450 cm-1 and 1770 cm-1 – 1050 cm-1) arise from the overlap of several stretching and bending vibrations (see Table S1 for detailed information), it is not possible to precisely separate and quantify the different components due to their proximity in the spectrum. To simplify and clarify spectral analysis, we also acquired the spectrum of nanoP3 that did not contain nitrogen by synthesizing nanoparticles in nitrogen-free C2H2/Ar plasmas (Figure 6a and 6b blue lines). The infrared spectrum of this


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nanoP3 formulation was characterized by absorption bands assigned to vibrations of OH, CH, C=O, C=C and C-H bonds (see Table S1 for detailed information).The incorporation of nitrogen in nanoP3 by synthesis in C2H2 /Ar / N2 plasmas was confirmed by a significant broadening of the absorption bands and occurrence of new additional peaks. New bands were observed due to vibrations of NH, C≡N, C=N, N-O, C-N (see Table S1). The detection of CN and NH bonds in the FTIR spectra demonstrates that the addition of non-polymerizable molecular gases (such as nitrogen) in the synthesis process provides means to straightforwardly modulate the chemical composition and surface properties of nanoP3. The CO, OH and NO bonds were formed upon exposure of nanoP3 to the atmosphere, likely driven by reaction of atmospheric oxygen with radicals formed in the plasma, and during synthesis by the incorporation of oxygen present at trace concentrations inside the plasma reactor.

Further quantification of the surface chemical composition with XPS revealed that the nitrogen fraction incorporated into as-synthesized nanoP3 was 33 % at a coupled power of 50 W, increasing to 37 % and 38 % at 75 W and 100 W respectively. The overall carbon relative elemental fraction was constant (≈ 63 %) over long-term storage of nanoP3 in air and at room temperature, up to one year (Figure 6c). However, we observed that the nitrogen fraction decreased to ≈ 23 % due to addition of surface oxygen of up to ≈ 13 % one-year post-synthesis. Deconvolution of high resolution XPS C 1s and N 1s core-shells (Figure 6d) revealed multiple contributions assigned to CC, CH, CN, COOH, NH, and NO hybridizations, further confirming the presence of surface functionalities (a detailed assignment of the C1-C4 and N1-N4 contributions is available in the supporting information file). FTIR and XPS measurements are consistent with our hypothesis that surface functional groups drive the charging of nanoP3 in solution. Increasing the rf coupled power


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increases the overall nitrogen atomic fraction and ultimately the total number of amine groups on the surface of nanoP3 in solution. Therefore, a higher solution pH is necessary to deprotonate the larger number of positively charged amine groups. Consequently, the isoelectric point of nanoP 3 occurs at progressively higher pH with increasing nitrogen content (i.e. nanoP 3 formulations produced at higher rf coupled power). In conclusion, the stability of nanoP 3 suspended in a solution is controlled by the solution pH at which their isoelectric point occurs, the latter driven by its surface chemistry, which is easily tuned by adjusting the plasma process parameters. Overall, correlations between OES in situ diagnostics and ex situ XPS and FTIR also suggest that radical molecules found in the plasma, such as CN, promote the formation of PCN and enhance the growth of nanoP3 through radical accretion. Further, our surface characterization suggests that nanoP3 are three-dimensional analogues of previously reported plasma-activated amorphous coatings

29, 32

.

These coatings, when applied to medical implants, were found to be well tolerated in vivo 33 and able to immobilize biomolecules in their active state via radical sites on their surface.

Since radical-chain polymerization growth 34 appears to play a significant role in the formation of nanoP3 (Figure 7a), we evaluated the presence of stable radicals within the nanoparticle structure using EPR spectroscopy. Unpaired electrons were detected within nanoP 3, as shown by the broad resonance peak (Figure 7b) around 348 mT (g factor = 2.003), and their kinetics are controlled by temperature-dependent diffusion towards the surface (Figure 7c). The radical volume density was ~ 3 x 1016 spins/cm3 measured 75 minutes post synthesis, decaying below 50 % of their initial value after 48 and 144 hours of storage in water at 24 ˚C and 4 ˚C respectively. The radical decay was slower for storage in air at 24 ˚C, with nanoP3 retaining 57 % of the initial radical density 240 hours post synthesis. FTIR measurements (Figure 7d) showed a significant increase in oxygen


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containing functional groups (e.g. stretching vibrations of OH and CO bonds), which were generated by peroxyl radicals formed in surface reactions of carbon-centered radicals with atmospheric oxygen. Surface oxidation was further enhanced either by producing nanoP 3 at higher base pressures (i.e. increased amount of residual oxygen in the plasma chamber) or by storing the particles in air for longer periods of time.

Linker-free and rapid one-step conjugation with bioactive cargo The presence of long-lived radicals within nanoP3 suggests the potential for direct immobilization of biomolecules by simple incubation

35-36

, giving nanoP3 an intrinsic advantage over competing

wet chemistry-based nanoparticle platforms. A radical based immobilization platform allows simultaneous attachment, without chemical intermediates, of cargo including ligands, imaging and therapeutic agents (Figure 8a). To exemplify and visualize the possibility of multifunctionalization, we immobilized three different IgG antibodies tagged with 40, 20 and 10 nm gold labels respectively and imaged under SEM, demonstrating single, double and triple antibody functionalization on a single nanoparticle (Figure 8b). To more quantitatively understand this potential, we then used representative conjugate classes with wide-ranging molecular weights and structures including an antibody (IgG, 150 kDa), enzyme (luciferase, 62 kDa), ribonucleic acid (small interfering RNA, 15 kDa), drug (paclitaxel, 1.3 kDa), fluorescent dye (cardiogreen, 0.78 kDa) and radical scavenger molecules (2,2-diphenyl-1-picrylhydrazyl, 0.39 kDa). We found that the loading capacity of nanoP3 was dependent on the available particle surface area, the molecular weight and geometry of the conjugate (Figure 8c). A full monolayer of bound conjugates was achieved by simple one-step incubation of nanoP3 with the cargo in aqueous solution and without


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intermediate chemical linkers. For example, the amount of drug (paclitaxel), imaging agent (ICG) and antibody (IgG) molecules bound to nanoP3 increased with their concentration in solution, saturating at a monolayer respectively with 2.3 μg, 2.9 μg and 0.8 μg per 10 9 nanoP3 (Figure 8d).

We then studied the binding kinetics associated with the simple nanoP 3 conjugation process by measuring the amount of bound cargo at different incubation time points and temperatures (Figure 8e). This was first demonstrated by incubating nanoP3 with sufficient ICG in solution necessary to saturate the nanoparticle surface at a monolayer, i.e. 1.9 x 10 6 ICG molecules on a single nanoP3 particle (≈ 200 nm). Following incubation, we washed the samples by ultracentrifugation and measured the absorbance spectra of the washes to quantify the remaining free ICG in the solution. We found that an incubation of the cargo with nanoP3 in water at 24 ˚C resulted in 90 % surface coverage within the first 30 seconds. The conjugation process at 4 ˚C was significantly slower, with 60 % of the nanoP3 surface covered with immobilized ICG after the same time-point.” Nevertheless, surface saturation of the nanoparticles was achieved in less than 5 minutes for both conditions. The simple and rapid incubation process in solution shows that nanoP3 provides a cost and time effective nanoparticle platform that does not require linker chemistry or long incubation periods. A long shelf-life and easy path to functionalization are also necessary requirements for a nanoparticle platform applicable for clinical use 3. We tested the binding efficiency of nanoP 3 stored for long periods of time and found that its ability to immobilize cargo remains virtually constant during the first 6 months of storage in air at room temperature (Figure 8f). We observed a decrease in the binding efficiency of 28 % for samples stored up to 16 months in the same conditions. However, it was still possible to fully saturate the surface of nanoP3 by increasing the concentration of ICG in solution. Extended shelf-life could potentially be achieved by employing


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alternative storage approaches, including low temperature, vacuum or another non-reactive environment. While the nanoP3 shelf life was exemplified here with ICG, we expect similar results with other types of molecular cargo since the immobilization properties of nanoP 3 are only limited by the available surface area and size of the cargo as shown in Figure 8c. We then tested the ability of nanoP3 to retain the activity of bound ICG with storage time in aqueous solution and compared with free ICG. Degradation of free ICG was observed by a 65 ± 2 % decrease of the absorption peak at 780 nm, 144 hours after reconstitution (Figure 8g), caused by the formation of leucoforms of the dye and driven by the saturation of double bonds in the compound chain 37. However, ICG bound to nanoP3 retained 91 ± 2 % of its initial activity during the same period. The absorbance spectrum of nanoP3-ICG constructs was red-shifted by 60 nm to 840 nm compared with free ICG (peak at 780 nm), further confirming the binding of ICG to the nanoP3 surface. Similar red-shift have been previously reported in various nanoparticle-ICG systems 38. These results show that in addition to robust immobilization of cargo, nanoP 3 can prolong the activity of some small (< 1 kDa) cargoes.

We also investigated the binding of nanoP3 to a highly-charged cargo, using a positively charged peptide sequence (CPKKKRKV). Initial incubations of nanoP 3 with the peptide at neutral pH did not result in the expected robust conjugation observed for more neutral cargo (Figure 8h). Increasing solution pH to 10 was required for binding to the highly positively charged peptide, consistent with the observed drop in nanoparticle zeta potential between pH 8 and 10 (Figure 5f). This demonstrated that manipulation of solution pH can be used to enhance the binding of charged ligands and potentially orient them on the surface should they have a dipole moment. We further demonstrated the robust binding of nanoP3 to conformationally sensitive molecules and compared


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with commercial gold (GNP) and polystyrene (PS) nanoparticles using luciferase as a probe (Figure 9). Luciferase is a bioluminescent enzyme, chosen to allow simultaneous study of functional activity as it is conformationally sensitive and inactive when unfolded

39 35

, .

Nanoparticles were incubated with luciferase, followed by three centrifugation washing steps after which the bioluminescence of the supernatant (Figure 9a) and the post-wash particles was recorded (Figure 9b). The assay demonstrated that nanoP3 provides a robust immobilization while preserving luciferase activity without any pre-functionalization with chemical cross-linking intermediates. Conversely, signal was detected in the first wash for gold nanoparticles, which require thiol mediated bonds to achieve covalent binding 40, and absent from polystyrene under all conditions due to suspected unfolding and associated loss of activity (Figure 9c).

NanoP3 interaction with cells We further investigated the interaction of nanoP 3 with multiple cell types. Penetration of the cell membrane, subsequent access to the cytoplasm and low toxicity are necessary requisites for nanoparticles to be useful in delivery of bioactive or therapeutic agents

41

. Positively charged

nanoparticle platforms including gold, quantum dots and dendrimers can penetrate the negatively charged cell membrane, but can leave behind membrane pores which increase cell death

42

.

Specialized nanoparticle shapes, including rods, needles and discs can also pass inside cells at a greater rate than traditional spherical architectures

43

. In the most recent studies, it is the protein

corona adsorbed to the nanoparticle surface which determines cellular interactions making the nature of surface interactions with serum proteins of critical importance 44.


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We observed that unfunctionalized ≈200 nm nanoP3 penetrated the membrane of human coronary artery endothelial cells (hCAECs) and accumulated in small clusters within the cytoplasm after 24 hours of incubation, observed using serial block face (SBF) SEM (Figure 10a). SBF SEM facilitates serial sectioning of single cells inside the scanning electron microscope, providing definitive evidence for the presence of nanoP3 inside the cell membrane. Reconstruction of multiple slices shows the clear presence of nanoP3, appearing as dark spheres outside of the nucleus (Movie S4). 3D rendering of the SBF SEM images best shows the distribution of nanoP 3 inside the cell membrane (Figure 10b). These results indicate that nanoP 3 readily cross the cell membrane without the requirement for surface functionalization with cell penetrating agents 1, in contrast to gold and polymer-based

46

45

platforms which benefit from peptide conjugation. While nanoP 3 are

positively charged at physiological pH it is likely that they rapidly conjugate serum proteins when added to the cell media, consistent with their binding behavior described above, and that these attached proteins drive interactions with the cell membrane. While further studies will determine the preferred pathway of cell internalization for differently sized nanoP3, we next sought to determine their cytocompatibility with various cell types.

Further nanoP3 incubation with hCAECs, epithelial MCF10A or human breast adenocarcinoma cells (MCF7) for up to 5 days did not significantly affect cell proliferation, with up to 3.1x108 particles per mm2 (Figure 10c). Rates of growth were marginally lower in HCAECs for all nanoparticle concentrations, somewhat variable for MCF10A with no clear trend for concentration and unchanged for MC7 compared to cell only controls. Cell morphology also appears unaffected


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after 3 and 5 days in culture (Figure S1), demonstrating that nanoP 3 were well tolerated in multiple cell types at high concentrations.

The addition of multiple functionalities to nanoparticles without the requirement for post synthesis modification and purification would break one of the critical impasses preventing timely clinical translation of new therapies 7. Transfer of multiple cargo into cells was demonstrated using paclitaxel-488, IgG-Cy5 and IgG-Cy7 as triple functionalized nanoP3 in MCF10A (Figure 10d), MCF7 and hCAECs (Figures S2 and S3). Confocal imaging showed the presence of paclitaxel (green), IgG-Cy5 (red) and IgG-Cy7 (orange) co-localized within the cell membrane. These data show that multiple cargo can be carried across the cell membrane when bound to nanoP 3, appearing to remain bound after entry. As we do not expect that the surface of nanoP 3 was fully saturated with cargo at the incubation concentrations used, the presence of additional serum proteins on the surface is likely to have played a role in interactions with the cell. While this paper demonstrates the retention of luciferase bioactivity bound to nanoP 3, consistent with our previous findings of conformation retention on plasma polymerized surfaces 32, retention of antibody/drug functionality once inside the cell was not determined in this first proof-of-concept experiment. Further studies are required to validate that intracellular activity can be retained. The current data set suggests that nanoP3 are effective carriers to transport ligands to the cytosol, but not the nucleus and that the covalent binding to the platform prevents cargo release once inside the cell. Future work will determine the utility of cleavable intermediates, well validated in other systems

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to facilitate

intracellular release and nuclear transport for payloads with this additional requirement.


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CONCLUSION The synthesis of multifunctional nanoparticles in a low temperature and low pressure plasma/gas phase offers a series of benefits over traditional multi-step wet-chemistry approaches. Here, we demonstrated that the physical and chemical properties of nanoP3, including aggregation, size distribution, charge and surface chemistry are modulated by appropriate choice of process parameters. NanoP3 retain a similar surface activity characteristic of thin film coatings synthesized by surface plasma polymerization. Functional radical groups activated in excess during the formation of initial PCN clusters (protoparticles), and active radical molecules diffusing to the particles during rapid cluster aggregation, are preserved within the nanoP3 carbon matrix. This unique radical-rich chemistry, allows for robust immobilization of cargo without the need for intermediate functionalization and purification steps, and combined with a long active shelf life, has great potential to overcome many of the major limitations of currently proposed nanoparticle platforms. The robust and versatile immobilization features of nanoP 3 were demonstrated here using a library of biomolecules of wide ranging structures, molecular weights and biological functions. Importantly, the biological activity and conformation of the molecules is preserved on the nanoP3, as suggested by the immobilization of the bioluminescent and conformationally sensitive enzyme luciferase. The exceptional surface of nanoP3 combined with their negligible cytotoxicity and intracellular trafficking properties are required features, paramount in facilitating the translation of nanoparticle-based therapy libraries into the commercial and clinical domain. As many of the favorable characteristics of nanoP3 are inherent to the fabrication process, we propose plasma polymerization in dusty plasmas as a nanoparticle synthesis route with valuable potential for broad clinical and commercial applications.


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METHODS NanoP3 synthesis and collection NanoP3 were synthesized in a cylindrical stainless steel capacitively coupled radio-frequency (rf) reactor. The reactor was pumped down to a base pressure of ~10-6 Torr using a dual dry vacuum pump system. Reactive gaseous mixtures of argon, nitrogen and acetylene were used to sustain the plasma discharge. The flow rate of each gas was individually controlled by Allicat Scientific mass flow controllers and the working pressure, measured by a full range gauge (PFEIFFER PKR251), was 150 mTorr prior to plasma generation. A butterfly-type valve was placed between the chamber and the pumping system and was adjusted to control the pumping efficiency and therefore the residence time of the species in the active plasma. Plasma discharges were generated and sustained by coupling the rf power, supplied by a 13.56 MHz (Eni OEM-6) frequency generator, to the top electrode using a purpose built matching network. The nanoP3 polystyrene collector vials (3.4 cm3) were placed in a second electrode (10 cm distant from the rf electrode). nanoP3 were resuspended directly from collector vials under sterile conditions in the tissue culture hood with RT-PCR Grade water (Life Technology). Optical emission spectroscopy diagnostics The synthesis of nanoP3 was monitored in situ using non-invasive optical emission spectroscopy. The radiation emitted by the plasma was captured by an optical fiber placed in line of sight with the plasma region ≈1 cm above thenanoP3 collector. High resolution spectra were obtained using an Acton SpectraPro 2750 spectrometer (Princeton Instruments, USA) equipped with a 1200 grooves mm-1 grating, resulting in a nominal resolution of 0.014 nm for an entrance slit opened at 50 𝜇m. Discharge emission spectra were captured by a PI-MAX (Princeton Instruments, USA)


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intensified charge-coupled device (ICCD) with a 1024 x 1024 pixel array optically coupled with the spectrometer’s exit plane. The ICCD exposure time was 300 𝜇s and 150 consecutive acquisitions were averaged to maximize the signal-to-noise ratio. The entire optical setup remained unchanged throughout the experiments. Measurements were performed to obtain the temporal profile of the intensities associated with specific radiative transitions involving several species in the plasma. Scanning and transmission electron microscopy SEM was performed with a Zeiss ULTRA Plus or a Zeiss Sigma HD FEG scanning electron microscope at acceleration voltages ranging 3 – 10 kV and a working distance between 3 – 12 mm. For TEM imaging, nanoP3 were collected on a 300-mesh grid with an ultrathin carbon film supported by a lacey carbon film (ProSciTech) placed inside the collector vials. Sample imaging was carried out in a JEOL 2100 TEM at an acceleration tension of 120 kV. For tomography, tilt series (±60˚) were acquired around a single axis using a 1˚ sampling increment. Tomography alignment and reconstruction was calculated using the Composer tomography package. Segmentation and rendering were created using Avizo 9.0.

Laser-light scattering for hydrodynamic size, concentration and zeta potential measurements The hydrodynamic size distribution and concentration of nanoP 3 in RT-PCR Grade water was measured in a NanoSight NS300 laser light scattering system. For each measurement, samples were introduced into the analysis chamber and allowed to flow at a constant flow rate and temperature (24˚ C). The trajectories of nanoP 3 in the chamber were visualized and tracked using


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a nanoparticle tracking and analysis system (NanoSight NTA 3.0). Data for each set of samples resulted from the continuous tracking (60 seconds periods) of the nanoP 3 trajectories and subsequent statistical analysis of five independent measurements. The zeta potential of nanoP 3 was measured within a pH range of 2 – 10 in disposable folded capillary cells (Malvern, DTS1070) using a Zetasizer Nano ZS (Malvern Instruments, Germany). Each sample was measured at a constant temperature of 24˚ C and the zeta potential was obtained from the average of three independent series of 11 measurements each. The quality of the measurements was monitored in real time and the data was analyzed using standard procedures using the software provided by the manufacturer. Electron spin resonance spectroscopy The detection of unpaired electrons in nanoP3 was carried out by means of ESR spectroscopy using a Bruker EMXplus X-band spectrometer. nanoP3 were suspended from the collector vials in RTPCR Grade water and transferred to quartz glass Suprasil flat cells with a sample volume capacity of 150 µL. Spectra were collected with a center field of 3490 G, sampling time of 90 ms, modulation amplitude of 3 G, modulation frequency of 105 Hz and microwave frequency and power of 9.8 GHz and 25 mW respectively. Conjugation of nanoP3 to various molecular cargo Goat anti-Rat IgG-Alexa647 (abcam, ab150159 from 0.5 to 3.5 µg/mL), Goat anti-Rabbit IgG-750 (abcam, ab175733 0.5 to 3.5 µg/mL), Goat anti-Rabbit-IgG-40 nm Gold (abcam, ab119180 at 2.5 µg/mL), Goat anti-Mouse-IgG-20 nm Gold (abcam, ab27242 at 2.5 µg/mL), Goat anti-Rat-IgG10 nm Gold (abcam, ab41512 at 2.5 µg/mL), 2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma Aldrich, from 0.5 to 10 µg/mL), Cardiogreen (Sigma Aldrich, from 0.5 to 10 µg/mL) and/or Paclitaxel-


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Oregon Green 488 (Invitrogen, P22310 from 0.5 to 3.5 µg/mL) were conjugated to nanoP 3 (109 nanoparticles/mL) in RT-PCR Grade water by simple incubation at 4°C for 1 hour, unless otherwise specified. Highly charged Cy5 labelled peptide sequences (CY5 – CPKKKRKV – NH2, Mimotopes at 2.5 µg/mL) were conjugated to nanoP3 (109 nanoparticles/mL) at pH 2, 7.4 and 10 (pH was adjusted in RT-PCR Grade water with HCl or NaOH). For all of the above molecules, the unbound cargo was washed three times by centrifugation at 16100 G for 5 minutes per wash. All washes were collected for absorbance and/or fluorescence measurements in order to calculate the amount of cargo bound to nanoP3. The amount of molecular cargo bound to nanoP3 following incubation was calculated using absorbance and/or fluorescence measurements of the nanoP 3– cargo samples and their corresponding washes (cargo = 2,2-diphenyl-1-picrylhydrazyl, cardiogreen, Paclitaxel-Oregon Green 488, CY5 – CPKKKRKV – NH2 and Goat anti-Rat IgGAlexa647). To calculate the number of cargo molecules bound to each nanoP 3 we first measured the absorbance/fluorescence of the remaining unbound cargo in the washes and interpolated the data to previously measured standard curves of the corresponding free molecular cargo. We then measured the absorbance/fluorescence of the nanoP3–cargo samples and subtracted the nanoP3 background signal to confirm that the sum of the signals of unbound cargo and cargo bound to nanoP3 matched the total amount of cargo initially added. Absorbance measurements were carried out via UV-VIS spectroscopy in a SHIMADZU UV-2550 spectrophotometer using disposable cuvettes and analyzed using UV Probe 2.33 software. The fluorescence intensity of samples and washes was measured using CLARIOstar microplate reader.


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Conjugation and activity of Luciferase on nanoP3 Luciferase from Photinus pyralis (Abcam, L9506, from 0.5 to 10 µg/mL) was conjugated to nanoP3 (109 nanoparticles/mL) in RT-PCR Grade water also by simple incubation at 4°C for 1 hour. Conjugation of Luciferase to 200nm Polystyrene (ThermoFisher Scientific; R200) and 200nm gold nanoparticles (Cytodiagnostics; G-150) were performed in an identical fashion. After incubation, samples were washed 3 times via centrifugation at 16100 G, 5 mins per wash, whilst the controls were washed 3 times per manufacturer’s recommendations. All wash supernatants were collected for detection and analysis of luminescence. After incubation and wash, we added D-Luciferin (Sigma-Aldrich; A1888) containing 70 mM ATP-Mg2+ to all samples and washes. The luminescence and luciferase activity was detected via IVIS system (IVIS Lumina LT, PerkinElmer) using an exposure time of 60 s.

Serial block face SEM sample preparation, mounting and imaging Human Coronary Artery Endothelial cells (hCAEC, passage 4-6, Cell Applications) were seeded at a cell density of 10000 cells/cm2 on circle glass cover-slips. Four hours after cell-seeding, 2.5 x 108 nanoparticles/mL in Human MesoEndo Endothelial growth media (Sigma-Aldrich) were added and left to incubate for 24 hours at 37°C in a 5% CO2 incubator. An equivalent volume of DMEM-serum free media without nanoparticles were added as a control. Samples were prepared for serial block face SEM (SBF SEM) using a modification of the protocol previously described by Deerinck et al47 (further information can be found in the supporting information file). Images were acquired using a variable pressure, field emission scanning electron microscope (Sigma VP, Carl Zeiss) equipped with a Gatan 3View 2XP. Backscattered electron images were collected at a fixed working distance of 4.4 mm using the following imaging parameters: 2.8 kV, 28 Pa, 30 μm


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aperture, 6000 x 12000 pixels, XY pixel size 6 nm, Z pixel size (slice thickness) 50 nm and pixel dwell time 1 μs.

Imaging of fluorescently labelled nanoP3 in cells hCAEC (human Coronary Artery Endothelial Cells, Cell Applications), MCF10A (Human Mammary Epithelial Cell line) and MCF7 cells (Cancerous Human Mammary Epithelial Cell line, Sigma-Aldrich) were seeded at a density of 10000 cells/cm2 on Lab-Tek glass chamber slides (LabTek). Four hours after cell-seeding, nanoP3 samples and controls were incubated with cells for 24 hours. The cells were then fixed with 3.7% paraformaldehyde for 10 minutes at room temperature and washed 3 times with PBS prior to F-actin and DAPI staining of cells. For F-actin staining, cells were permeabilized with 0.01% triton-X 100 (Sigma-Aldrich, Cat) in PBS for 5 minutes. The samples were then washed 3 times in PBS, followed by incubation with Actin Red 555 ReadyProbes reagent (Life technology) for 15 minutes, as per manufacturer recommendations. The samples were then washed 3 times in PBS. For DAPI staining of cell nuclei, cells were incubated with 1:1000 DAPI (SAPBIO) in PBS for 5 minutes at room temperature. The samples were washed 3 times in PBS prior to mounting with an aqueous fluorescent-shield mounting medium (Dako). After cover-slipping, the images were taken within 24 hours under a fluorescent microscope.

Cell proliferation assay

NanoP3 cytotoxicity was evaluated for 3 different cells types: 1) human Coronary Artery Endothelial Cells (hCAEC, passage 4, Cell Applications); 2) Human Mammary Epithelial Cell line (MCF10A, passage 20) and 3) Cancerous Human Mammary Epithelial Cell line (MCF7, passage 15, Sigma-Aldrich). Cells were trypsinized and 5000 cells/cm2 were seeded in a flat-bottomed 24-


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well culture plate and incubated at 37°C in a 5% CO2 incubator. Four hours after cell seeding, serial dilutions of nanoP3 (from 102 nanoparticles/well to 109 nanoparticles/well), were resuspended in DMEM serum-free media, and then added into each well. After 3 and 5 days of incubation, the cell viability was evaluated using alamarBlue (Life Technologies). The alamar Blue reagent was added to each well as per manufacturer’s recommendations and incubated for 2 hours (for hCAEC and MCF10A), and 3 hours (for MCF7). The resorufin dye catalyzed by viable cells was quantified by measuring the fluorescence at Ex/Em 530-560/590 nm on a microplate reader (Manufacturer). Untreated cells (DMEM serum-free media without nanoP3) and cells treated with doxorubicin (500nM, Sigma-Aldrich) were used as controls.

Statistical analysis Data are expressed as mean ± s.d. Data were analyzed using one-way ANOVA analysis of variance (Tukey’s multiple comparison groups). P-Value < 0.05 were considered significant.


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FIGURES

Figure 1. Schematic diagram showing formation, growth and collection of nanoP3 in the plasma/gas phase. The plasma reactive chemistry determines the formation and nucleation of primary nanoP3 protoparticles (or clusters) in phase 1 that rapidly aggregate to form spherical nanoP3 (phase 2). When nanoP3 reach a critical size, gravitational and ion drag forces push them out of the plasma (phase 3), enabling their collection in electrically biased collector vials. An estimate of the time taken for each phase is indicated in seconds. Insets show images of the plasma before and during nanocluster aggregation and a SEM micrograph of nanoP 3 as collected. Scale bar = 200 nm.


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Figure 2. (a) Time series of the discharge optical emission spectra (386 – 396 nm) during the cluster (protoparticle) formation and growth phases. (b) Time-evolution of the CN, N2+ and N2 molecular rotational-vibrational band emission intensities during nanoP 3 formation shows the plasma emission intensity cycles over the three phases of nanoP 3 formation, growth and collection. The rapid nanocluster aggregation coincides with an increase of both collected nanoP 3 and discharge emission intensity. (c) nanoP3 synthesis in a dusty plasma is a continuous cyclic process as shown here by 30 consecutive oscillation cycles, where nanoP3 is continuously formed and subsequently collected at the end of each cycle.


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Figure 3. The period of the nanoP3 formation cycles is modulated by plasma parameters, including (a) pressure, (b) acetylene flow rate and rf power. (c) Modulation of the nanoP3 yield showed a non-linear behavior with the rf power and acetylene flow rate, maximizing at 14 g/hr.m2 for 100 W and 6 sccm, which is compatible with commercial production rates.


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Figure 4. (a) Representative HR-TEM images of as synthesized nanoP3 acquired at different stage tilting angles. b) Various z-stack images from the resulting electron tomogram obtained by alignment and computer reconstruction of the 2D HR-TEM images in (a). (c) Representative volume rendering, (d) SEM and (e) HR-TEM of as synthesized nanoP3. Results show that nanoP3 is formed by the aggregation of PCN (protoparticles) that assemble to form a compacted spherical-like structure. (f) The FFT the HR-TEM image (inset in Figure 4e), features a diffuse halo with the absence of rings, typical of amorphous materials. Scale bars are 100 nm in (a) and (b), 50 nm in (c) and (d), 20 nm in (e) and 10 nm in inset in (e).


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Figure 5. Size distribution analysis by (a) SEM (b) and dynamic light scattering (DLS) shows that nanoP3 size is modulated by the applied rf power at the maximum yields (3 sccm at 50 W and 75W and 6 sccm at 100W). For comparison, the stars and arrows indicate the average particle size measured by the complementary techniques SEM (a) and DLS (b). (c) When compared to assynthesized and dry nanoP3 (SEM), the size of nanoP3 increased upon dispersion in aqueous solution (DLS) due to the formation of an ionic double layer. (d) Hydrodynamic median size of nanoP3 dispersed in PCR grade water as a function of the solution pH for the maximum yield rates. The size increased significantly in mildly alkaline media for all formulations due to particle aggregation. nanoP3 aggregation was reversed at higher pH for formulations prepared at 50 W and 75 W. (e) The polydispersity index (PDI) increases with the solution pH, which is consistent with particle aggregation. (f) 𝜁-potential measurements of nanoP3 show that the surface charge of the nanoparticles can be significantly modulated in the pH range 2 – 10. All samples were positively charged and stably dispersed in aqueous solution (i.e. 𝜁 > 30 mV) at physiological pH. The instability region ( -30 mV < 𝜁 < 30 mV) is shown in grey.


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Figure 6. Comparison of the FTIR (a) and XPS (b) survey spectra of nanoparticles formed in C 2H2/Ar and C2H2/Ar/N2 plasma discharges. (c) Fractional carbon, nitrogen and oxygen content on nanoP 3 at various time points up to 1-year post synthesis. (d) Deconvolution of the C 1s and N 1s XPS core levels of nanoP3 ([C]/[N] = 2). Taken together, these data suggest the incorporation of amines and carboxyl surface moieties in nanoP3 simply by adding N2 in the gaseous mixture and by exposing nanoP3 to air respectively. Samples were synthesized at 150 mTorr, 50 W and acetylene flow rate of 2 sccm.


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Figure 7. (a) Schematic diagram showing the diffusion of radical molecules, ions and electrons towards nanoP3 during their synthesis in a reactive dusty plasma. (b) EPR spectroscopy confirms the presence of long-lived radicals in nanoP3 after synthesis, shown here by a broad resonance peak centered around 348 mT. (c) Kinetics of radicals on nanoP 3 shows that radical diffusion is temperature-activated and slower when particles are dry-stored. (d) Absorbance ratios between stretching vibrations of OH (3500 cm-1) and CO (1700 cm-1) to aliphatic CH (2900 cm-1) on nanoP3 at various time-points post synthesis and two base pressures. NanoP3 samples were synthesized at 150 mTorr, 50 W and acetylene flow rate of 2 sccm.


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Figure 8. (a) Schematic diagram showing the simple and rapid nanoP 3 incubation process in solution with targeting ligands, proteins, imaging agents or small molecules, resulting in robust surface functionalization. (b) Exemplifying the utility of this approach, multiple functionalities can be added to the surface of nanoP3 by varying the incubation solution. SEM imaging demonstrates single (40 nm IgG gold label - blue), double (40 nm and 20 nm IgG gold labels – blue and magenta respectively) and triple (40 nm, 20 nm and 10 nm IgG gold labels – blue, magenta and green respectively) functionalization of a single nanoparticle. Scale bar is 60 nm. (c) Number of cargo molecules per unit surface area of nanoP3 at a monolayer for various cargo molecular weights, structures and biological functions. The total number of molecules bound to the nanoP3 surface is


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ultimately dependent on ratio between the nanoP 3 surface area and the molecular weight of the cargo. (d) The total cargo mass bound to nanoP3 increases with the mass added to the solution during the incubation process. Exemplification with a drug (paclitaxel), imaging agent (cardiogreen) and targeting ligand (IgG) demonstrated surface saturation at 2.3 μg, 2.9 μg and 0.8 μg per 109 nanoparticles respectively. (e) Binding kinetics experiments, exemplified here with cardiogreen, show that 90% of the total cargo was immobilized on the surface of nanoP 3 within the first 30 seconds after incubation at 24 ˚C, while incubation at lower temperature (4 ˚C) resulted in a 60 % binding for the same time point. Surface saturation was achieved in less than 5 minutes after incubation for both conditions. The inset shows the same data representing time in a log scale and the dashed line represents the point where 100% surface coverage occurs at a monolayer. (f) The long shelf-life of nanoP3 was demonstrated by their ability to bind ICG up to 6 months of storage in air and room temperature, decreasing by 28% after 16 months. (g) VIS/NIR profile of free ICG and ICG conjugated nanoP3 shows that degradation of small cargo (< 1000 Da) is significantly delayed once immobilized to nanoP3. The binding of ICG to the nanoP3 surface was confirmed by a 60-nm red-shift of the absorbance peak from 780 nm to 840 nm compared with free ICG in solution. (h) The binding ability of nanoP3 to highly positively charged cargo is exemplified with a highly-charged peptide sequence at different pH. Binding to nanoP 3 was only achieved at higher pH, consistent with zeta potential measurements. NanoP3 samples were synthesized at 150 mTorr, 50 W and acetylene flow rate of 2 sccm.


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Figure 9. (a) We compared conjugation with luciferase to commercial gold (GNP) and polystyrene (PS) nanoparticles. Following multiple washes, luciferase bioluminescence, requiring both binding and bioactivity was quantified. Evaluation of the supernatant from the first wash step showed a significant presence of luciferase for the GNP sample, suggesting non-binding of the enzyme to the GNPs. (b) Whilst in the final post-wash samples, only nanoP3 showed a positive signal, indicating retention of bioactive luciferase on the surface of these nanoparticles. The absence of signal for PS suggests unfolding of the enzyme. The bioluminescence signal is shown in triplicates for each sample at the top of its corresponding data bar. (c) Schematic diagram to demonstrate the robust binding facilitated by nanoP3.


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Figure 10. (a) 3View SEM demonstrates nanoP3 inside the cell membrane of human coronary artery endothelial cells, appearing as dark spheres (red arrows) outside of the nucleus (N). Scale bar = 16 μm. (b) 3D reconstruction of the 3View sections shows a homogenous distribution of nanoP3 (red) in the cytoplasm (green). The cell nucleus is shown in blue. (c) Cytotoxicity of nanoP 3 was evaluated in 3 different cell types (i) endothelial cells (hCAECs), (ii) MCF10A, and (iii) MCF7. After 3 and 5 days incubation at increasing nanoP 3 concentrations (up to 109 particles/3.2mm2), no significant reduction in viability was detected and proliferation rates appeared comparable to cell only controls. Doxorubicin (dox) was used as a negative control for cell growth. (d) nanoP3 co-functionalized with paclitaxel (green), IgG-Cy7 (red) and IgG-Cy5 (orange) entered MCF7 cells following incubation, accumulating in the cytoplasm. Scale bar = 20 μm. NanoP3 samples were synthesized at 150 mTorr, 50 W and acetylene flow rate of 2 sccm.


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ASSOCIATED CONTENT Supporting Information. FTIR and XPS detailed methods and peak assignment, SEM SBF extended methods, cell proliferation and morphology upon incubation with nanoP 3, fluorescent microscopy showing internalization on triple functionalized nanoP 3 in hCAEC, MCF10A and MCF7 cell groups. The supporting information is available free of charge on the ACS Publications website.

Corresponding Authors

* Miguel Santos: Tel: +61282088928; Email: [email protected]. * Steven Wise: Tel: +61282088928; Email: [email protected]. * Marcela Bilek: Tel: +61293516079; Email: [email protected].

Author Contributions S.W. and M.S. conceived the overall idea and initial research plan. M.S. designed synthesis, in situ monitoring and collection methods for nanoP 3. P.M., M.S. and E.F. designed and conducted experiments, supported by A.C., J.H., R.T., B.L., C.H. and A.W., M.S., S.W., M.B., P.M., E.F. and A.W. analyzed data. M.H. carried out serial block face SEM. M.S., S.W. and M.B. wrote the paper. S.W. and M.B. supervised the project. All authors discussed the results and commented on the final version of the manuscript. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally.


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Acknowledgments The authors would like to thank E. Brackenreg, the James N Kirby Foundation, Optiver Asia Pacific and the Heart Research Institute for funding support. The authors also acknowledge the facilities as well as scientific and technical assistance at the Australian Centre for Microscopy and Microanalysis at the University of Sydney. Special thanks to Dr. Patrick Trimby and Dr. Hongwei Liu for their kind assistance with SEM and TEM imaging. Funding sources This work was funded by the Heart Research Institute and donations from E. Brackenberg, the James N Kirby Foundation and Optiver Asia Pacific.

REFERENCES

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Table of contents graphic (84mm x 36mm – 600dpi)


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Figure 1. Schematic diagram showing formation, growth and collection of nanoP3 in the plasma/gas phase. The plasma reactive chemistry determines the formation and nucleation of primary nanoP3 protoparticles (or clusters) in phase 1 that rapidly aggregate to form spherical nanoP3 (phase 2). When nanoP3 reach a critical size, gravitational and ion drag forces push them out of the plasma (phase 3), enabling their collection in electrically biased collector vials. The time taken for each phase is indicated in seconds. Insets show images of the plasma before and during nanocluster aggregation and a SEM micrograph of nanoP3 as collected. Scale bar = 200 nm. 334x134mm (300 x 300 DPI)


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Figure 2. (a) Time series of the discharge optical emission spectra (386 – 396 nm) during the cluster (protoparticle) formation and growth phases. (b) Time-evolution of the CN, N2+ and N2 molecular rotational-vibrational band emission intensities during nanoP3 formation shows the plasma emission intensity cycles over the three phases of nanoP3 formation, growth and collection. The rapid nanocluster aggregation coincides with an increase of both collected nanoP3 and discharge emission intensity. (c) nanoP3 synthesis in a dusty plasma is a continuous cyclic process as shown here by 30 consecutive oscillation cycles, where nanoP3 is continuously formed and subsequently collected at the end of each cycle. 122x314mm (300 x 300 DPI)



Figure 3. The period of the nanoP3 formation cycles is modulated by plasma parameters, including (a) pressure, (b) acetylene flow rate and rf power. (c) Modulation of the nanoP3 yield showed a non-linear behavior with the rf power and acetylene flow rate, maximizing at 14 g/hr.m2 for 100 W and 6 sccm, which is compatible with commercial production rates. 67x181mm (300 x 300 DPI)


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Figure 4. (a) Representative HR-TEM images of as synthesized nanoP3 acquired at different stage tilting angles. b) Various z-stack images from the resulting electron tomogram obtained by alignment and computer reconstruction of the 2D HR-TEM images in (a). (c) Representative volume rendering, (d) SEM and (e) HR-TEM of as synthesized nanoP3. Results show that nanoP3 is formed by the aggregation of PCN (protoparticles) that assemble to form a compacted spherical-like structure. (f) The FFT the HR-TEM image (inset in Figure 4e), features a diffuse halo with the absence of rings, typical of amorphous materials. Scale bars are 100 nm in (a) and (b), 50 nm in (c) and (d), 20 nm in (e) and 10 nm in inset in (e). 294x168mm (300 x 300 DPI)



Figure 5. Size distribution analysis by (a) SEM (b) and dynamic light scattering (DLS) shows that nanoP3 size is modulated by the applied rf power at the maximum yields (3 sccm at 50 W and 75W and 6 sccm at 100W). For comparison, the stars and arrows indicate the average particle size measured by the complementary techniques SEM (a) and DLS (b). (c) When compared to as-synthesized and dry nanoP3 (SEM), the size of nanoP3 increased upon dispersion in aqueous solution (DLS) due to the formation of an ionic double layer. (d) Hydrodynamic median size of nanoP3 dispersed in PCR grade water as a function of the solution pH for the maximum yield rates. The size increased significantly in mildly alkaline media for all formulations due to particle aggregation. nanoP3 aggregation was reversed at higher pH for formulations prepared at 50 W and 75 W. (e) The polydispersity index (PDI) increases with the solution pH, which is consistent with particle aggregation. (f) ߞ-potential measurements of nanoP3 show that the surface charge of the nanoparticles can be significantly modulated in the pH range 2 – 10. All samples were positively charged and stably dispersed in aqueous solution (i.e. ߞ > 30 mV) at physiological pH. The instability region ( -30 mV < ߞ < 30 mV) is shown in grey. 343x214mm (300 x 300 DPI)


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Figure 6. Comparison of the FTIR (a) and XPS (b) survey spectra of nanoparticles formed in C2H2/Ar and C2H2/Ar/N2 plasma discharges. (c) Fractional carbon, nitrogen and oxygen content on nanoP3 at various time points up to 1-year post synthesis. (d) Deconvolution of the C 1s and N 1s XPS core levels of nanoP3 ([C]/[N] = 2). Taken together, these data suggest the incorporation of amines and carboxyl surface moieties in nanoP3 simply by adding N2 in the gaseous mixture and by exposing nanoP3 to air respectively. Samples were synthesized at 150 mTorr, 50 W and acetylene flow rate of 2 sccm. 278x210mm (300 x 300 DPI)



Figure 7. (a) Schematic diagram showing the diffusion of radical molecules, ions and electrons towards nanoP3 during their synthesis in a reactive dusty plasma. (b) EPR spectroscopy confirms the presence of long-lived radicals in nanoP3 after synthesis, shown here by a broad resonance peak centered around 348 mT. (c) Kinetics of radicals on nanoP3 shows that radical diffusion is temperature-activated and slower when particles are dry-stored. (d) Absorbance ratios between stretching vibrations of OH (3500 cm-1) and CO (1700 cm-1) to aliphatic CH (2900 cm-1) on nanoP3 at various time-points post synthesis and two base pressures. NanoP3 samples were synthesized at 150 mTorr, 50 W and acetylene flow rate of 2 sccm. 237x207mm (300 x 300 DPI)


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Figure 8. (a) Schematic diagram showing the simple and rapid nanoP3 incubation process in solution with targeting ligands, proteins, imaging agents or small molecules, resulting in robust surface functionalization. (b) Exemplifying the utility of this approach, multiple functionalities can be added to the surface of nanoP3 by varying the incubation solution. SEM imaging demonstrates single (40 nm IgG gold label - blue), double (40 nm and 20 nm IgG gold labels – blue and magenta respectively) and triple (40 nm, 20 nm and 10 nm IgG gold labels – blue, magenta and green respectively) functionalization of a single nanoparticle. Scale bar is 60 nm. (c) Number of cargo molecules per unit surface area of nanoP3 at a monolayer for various cargo molecular weights, structures and biological functions. The total number of molecules bound to the nanoP3 surface is ultimately dependent on ratio between the nanoP3 surface area and the molecular weight of the cargo. (d) The total cargo mass bound to nanoP3 increases with the mass added to the solution during the incubation process. Exemplification with a drug (paclitaxel), imaging agent (cardiogreen) and targeting ligand (IgG) demonstrated surface saturation at 2.3 µg, 2.9 µg and 0.8 µg per 109 nanoparticles respectively. (e) Binding kinetics experiments, exemplified here with cardiogreen, show that 90% of the total cargo was immobilized on the surface of nanoP3 within the first 30 seconds after incubation at 24 ˚C, while incubation at lower temperature (4 ˚C) resulted in a 60 % binding for the same time point. Surface saturation was achieved in less than 5 minutes after incubation for both conditions. The inset shows the same data representing time in a log scale and the dashed line represents the point where 100% surface coverage occurs at a monolayer. (f) The long shelf-life of nanoP3 was demonstrated by their ability to bind ICG up to 6 months of storage in air and room temperature, decreasing by 28% after 16 months. (g) VIS/NIR profile of free ICG and ICG conjugated nanoP3 shows that degradation of small cargo (< 1000 Da) is significantly delayed once immobilized to nanoP3. The binding of ICG to the nanoP3 surface was confirmed by a 60-nm red-shift of the absorbance peak from 780 nm to 840 nm compared with free ICG in solution. (h) The binding ability of nanoP3 to highly positively charged cargo is exemplified with a highly-charged



peptide sequence at different pH. Binding to nanoP3 was only achieved at higher pH, consistent with zeta potential measurements. NanoP3 samples were synthesized at 150 mTorr, 50 W and acetylene flow rate of 2 sccm. 326x302mm (300 x 300 DPI)


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Figure 9. (a) We compared conjugation with luciferase to commercial gold (GNP) and polystyrene (PS) nanoparticles. Following multiple washes, luciferase bioluminescence, requiring both binding and bioactivity was quantified. Evaluation of the supernatant from the first wash step showed a significant presence of luciferase for the GNP sample, suggesting non-binding of the enzyme to the GNPs. (b) Whilst in the final post-wash samples, only nanoP3 showed a positive signal, indicating retention of bioactive luciferase on the surface of these nanoparticles. The absence of signal for PS suggests unfolding of the enzyme. The bioluminescence signal is shown in triplicates for each sample at the top of its corresponding data bar. (c) Schematic diagram to demonstrate the robust binding facilitated by nanoP3. 219x215mm (300 x 300 DPI)



Figure 10. (a) 3View SEM demonstrates nanoP3 inside the cell membrane of human coronary artery endothelial cells, appearing as dark spheres (red arrows) outside of the nucleus (N). Scale bar = 16 µm. (b) 3D reconstruction of the 3View sections shows a homogenous distribution of nanoP3 (red) in the cytoplasm (green). The cell nucleus is shown in blue. (c) Cytotoxicity of nanoP3 was evaluated in 3 different cell types (i) endothelial cells (hCAECs), (ii) MCF10A, and (iii) MCF7. After 3 and 5 days incubation at increasing nanoP3 concentrations (up to 109 particles/3.2mm2), no significant reduction in viability was detected and proliferation rates appeared comparable to cell only controls. Doxorubicin (dox) was used as a negative control for cell growth. (d) nanoP3 co-functionalized with paclitaxel (green), IgG-Cy7 (red) and IgG-Cy5 (orange) entered MCF7 cells following incubation, accumulating in the cytoplasm. Scale bar = 20 µm. NanoP3 samples were synthesized at 150 mTorr, 50 W and acetylene flow rate of 2 sccm. 220x258mm (300 x 300 DPI)


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