Name: evaluation of antimicrobial activities of nanoparticles and nanostructured surfaces in vitro
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The authors appreciate the financial support from the U.S. National Science Foundation (NSF CBET award 1512764 and NSF PIRE 1545852), the National Institutes of Health (NIH NIDCR 1R03DE028631), the University of California (UC) Regents Faculty Development Fellowship, the Committee on Research Seed Grant (Huinan Liu), and the UC-Riverside Graduate Research Mentorship Program Grant awarded to Patricia Holt-Torres. The authors appreciate the assistance provided by the Central Facility for Advanced Microscopy and Microanalysis (CFAMM) at UC-Riverside for the use of SEM/EDS and Dr. Perry Cheung for the use of XRD. The authors would also like to thank Morgan Elizabeth Nator and Samhitha Tumkur for their assistance with the experiments and data analyses. Any opinions, findings, conclusions, or recommendations expressed in this article are those of the authors and do not necessarily reflect the views of the National Science Foundation or the National Institutes of Health.

To present the direct co-culture and direct exposure methods, we use magnesium oxide nanoparticles (nMgO) as a model material to demonstrate bacterial interactions. To present the direct culture and focused-contact exposure methods, we use an Mg alloy with nanostructured surfaces as examples.
1. Sterilization of nanomaterials
NOTE: All the nanomaterials must be sterilized or disinfected prior to microbial culture. The methods that can be used include ...

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We have presented four in vitro methods (A-D) to characterize the antibacterial activities of nanoparticles and nanostructured surfaces. While each of these methods quantifies bacterial growth and viability over time in response to nanomaterials, some variation exists in the methods used to measure the initial bacterial seeding density, growth, and viability over time. Three of these methods, the direct co-culture method (A)17, the direct culture method (C)14, and ...

In the US alone, 1.7 million individuals develop a hospital-acquired infection (HAI) annually, with one in every 17 of these infections resulting in death1. In addition, it is estimated that the treatment costs for HAIs range from $28 billion to $45 billion annually1,2. These HAIs are predominated by methicillin-resistant Staphylococcus aureus (MRSA)3,4 and Pseudomonas aeruginosa4, which are commonly isolated from chronic wound infections and usually require extensive treatment and time to produce a favorable patient outcome.
Over the past several decades, multiple antibiotic classes have been developed to treat infections related to these and other pathogenic bacteria. For example, rifamycin analogs have been used to treat MRSA, other gram-positive and gram-negative infections, and Mycobacterium spp. infections5. In the 1990s, to effectively treat an increasing number of M. tuberculosis infections, additional drugs were combined with rifamycin analogs to increase their effectiveness. However, approximately 5% of M. tuberculosis cases remain resistant torifampicin5,6, and there is increasing concern regarding multi-drug resistant bacteria7. Currently, the use of antibiotics alone may not be sufficient in the treatment of HAIs, and this has provoked an ongoing search for alternative antimicrobial therapies1.
Heavy metals, such as silver (Ag)8,9,10 and gold (Au)11, and ceramics, such as titanium dioxide (TiO2)12 and zinc oxide (ZnO)13, in nanoparticle (NP) form (AgNP, AuNP, TiO2NP, and ZnONP, respectively) have been examined for their antimicrobial activities and have been identified as potential antibiotic alternatives. In addition, bioresorbable materials, such as magnesium alloys (Mg alloys)14,15,16, magnesium oxide nanoparticles17,18,19,20,21, and magnesium hydroxide nanoparticles [nMgO and nMg(OH)2, respectively]22,23,24, have also been examined. However, the previous antimicrobial studies of nanoparticles used inconsistent materials and research methods, resulting in data that are difficult or impossible to compare and are sometimes contradictory in nature18,19. For example, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of silver nanoparticles varied significantly in different studies. Ipe et al.25 evaluated the antibacterial activities of AgNPs with an average particle size of ~26 nm to determine the MICs against gram-positive and gram-negative bacteria. The identified MICs for P. aeruginosa, E. coli, S. aureus,&#160;and MRSA were 2 &#181;g/mL, 5 &#181;g/mL, 10 &#181;g/mL, and 10 &#181;g/mL, respectively. In contrast, Parvekar et al.26 evaluated AgNPs with an average particle size of 5 nm. In this instance, the AgNP MIC and a MBC of 0.625 mg/mL were found to be effective against S. aureus. In addition, Loo et al.27 evaluated AgNPs with a size of 4.06 nm. When E. coli was exposed to these nanoparticles, the MIC and MBC were reported at 7.8 &#181;g/mL. Finally, Ali et al.28 investigated the antibacterial properties of spherical AgNPs with an average size of 18 nm. When P. aeruginosa, E. coli, and MRSA were exposed to these nanoparticles, the MIC was identified at 27 &#181;g/mL, 36 &#181;g/mL, 27 &#181;g/mL, and 36 &#181;g/mL, respectively, and the MBC was identified at 36 &#181;g/mL, 42 &#181;g/mL, and 30 &#181;g/mL, respectively.
Although the antibacterial activity of nanoparticles has been extensively studied and reported during recent decades, there is no standard for the materials and research methods used to allow for direct comparisons across studies. For this reason, we present two methods, the direct co-culture method (method A), and the direct exposure method (method B), to&#160;characterize and compare the antimicrobial activities of nanoparticles while keeping the materials and methods consistent.
In addition to nanoparticles, nanostructured surfaces have also been examined for antibacterial activities. These include carbon-based materials, such as graphene nanosheets, carbon nanotubes, and graphite29, as well as pure Mg and Mg alloys. Each of these materials has exhibited at least one antibacterial mechanism, including physical damage imposed on cell membranes by carbon-based materials and damage to metabolic processes or DNA through the release of reactive oxygen species (ROS) when Mg degrades. In addition, when zinc (Zn) and calcium (Ca) are combined in the formation of Mg alloys, the refinement of the Mg matrix grain size is enhanced, which leads to a reduction in bacterial adhesion to substrate surfaces in comparison to Mg-only samples14. To demonstrate antibacterial activity, we present the direct culture method (method C), which determines bacterial adhesion on and around nanostructured materials over time through the quantification of bacterial colony-forming units (CFUs) with direct and indirect surface contact.
The geometry of nanostructures on surfaces, including the size, shape, and orientation, could influence the bactericidal activities of materials. For example, Lin et al.16 fabricated different nanostructured MgO layers on the surfaces of Mg substrates through anodization and electrophoretic deposition (EPD). After a period of exposure to the nanostructured surface in vitro, the growth of S. aureus was substantially reduced in comparison to non-treated Mg. This indicated a greater potency of the nanostructured surface against bacterial adhesion versus the nontreated metallic Mg surface. To reveal the different mechanisms of the antibacterial properties of various nanostructured surfaces, a focused-contact exposure method (method D) that determines the cell-surface interactions within the area of interest is discussed in this article.
The objective of this article is to present four in vitro methods that are applicable to different nanoparticles, nanostructured surfaces, and microbial species. We discuss key considerations for each method to produce consistent, reproducible data for comparability. Specifically, the direct co-culture method17 and direct exposure method are used for examining the antimicrobial properties of nanoparticles. Through the direct co-culture method, the minimum inhibitory and minimum bactericidal concentrations (MIC and MBC90-99.99, respectively)&#160;can be determined for individual species, and the most potent concentration (MPC) can be determined for multiple species. Through the direct exposure method, the bacteriostatic or bactericidal effects of nanoparticles at minimum inhibitory concentrations can be characterized by real-time optical density readings over time. The direct culture14 method is suitable for examining bacteria directly and indirectly in contact with nanostructured surfaces. Finally, the focused-contact exposure16 method is presented to examine the antibacterial activity of a specific area on a nanostructured surface through the direct application of bacteria and the characterization of bacterial growth at the cell-nanostructure interface. This method is modified from the Japanese Industrial Standard JIS Z 2801:200016, and is intended to focus on microbe-surface interactions and exclude the effects of bulk sample degradation in microbial culture on antimicrobial activities.

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			<td>1.5 mL microcentrifuge tube</td>
			<td>Milipore Sigma</td>
			<td>Z336777</td>
			<td></td>
		</tr>
		<tr>
			<td>80 L NTRL Certified Convection Drying Oven&#160;</td>
			<td>MTI Corporation</td>
			<td>BPG-7082</td>
			<td>https://www.mtixtl.com/BPG-7082.aspx</td>
		</tr>
		<tr>
			<td>(hydroxymethyl) aminomethane buffer pH 8.5; Tris buffer&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>42457</td>
			<td></td>
		</tr>
		<tr>
			<td>AnaSpec&#160;THIOFLAVIN T ULTRAPURE GRADE</td>
			<td>Fisher Scientific</td>
			<td>50-850-291</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron-multiplying charge-coupled device digital camera&#160;</td>
			<td>Hamamatsu</td>
			<td>C9100-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15 mL conical tubes</td>
			<td>Fisher Scientific</td>
			<td>14-959-49B</td>
			<td></td>
		</tr>
		<tr>
			<td>Gluteraldehyde</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>G5882</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Brightline, Hausser Scientific</td>
			<td>1492</td>
			<td></td>
		</tr>
		<tr>
			<td>Inductively coupled plasma - optical emission spectrometry (ICP-OES)</td>
			<td>PerkinElmer</td>
			<td>8000</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverse microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Bertani Broth</td>
			<td>Sigma Life Science&#160;</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Bertani Broth + agar</td>
			<td>Sigma Life Science&#160;</td>
			<td>L2897</td>
			<td></td>
		</tr>
		<tr>
			<td>MacroTube 5.0&#160;&#160;</td>
			<td>Benchmark Scientific</td>
			<td>C1005-T5-ST</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium oxide nanoparticles</td>
			<td>US Research Nanomaterials, Inc</td>
			<td>Stock #:&#160;US3310&#160;&#160;&#160;M</td>
			<td>MgO, 99+%, 20 nm</td>
		</tr>
		<tr>
			<td>MS Semi-Micro Balance</td>
			<td>Mettler Toledo</td>
			<td>MS105D</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose paper</td>
			<td>Fisherbrand</td>
			<td>09-801A</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-tissue treated 12-well polystyrene plate</td>
			<td>Falcon Corning Brand&#160;</td>
			<td>351143</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-tissue treated 48-well polystyrene plate</td>
			<td>Falcon Corning Brand&#160;</td>
			<td>351178</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-tissue treated 96-well polystyrene plate</td>
			<td>Falcon Corning Brand&#160;</td>
			<td>351172</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 100 mm</td>
			<td>VWR</td>
			<td>470210-568</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish, 15 mm</td>
			<td>Fisherbrand</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>VWR</td>
			<td>SP70P</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscopy (SEM)</td>
			<td>TESCAN&#160;</td>
			<td>Vega3 SBH</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>VWR</td>
			<td>97043-936</td>
			<td></td>
		</tr>
		<tr>
			<td>Table top centrifuge</td>
			<td>Fisher Scientific</td>
			<td>accuSpin Micro 17</td>
			<td></td>
		</tr>
		<tr>
			<td>Table top centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>Centrifuge 5430</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic Soy Agar</td>
			<td>MP</td>
			<td>1010617</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic Soy Broth</td>
			<td>Sigma-Aldrich</td>
			<td>22092-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Vis spectrophotometer&#160;</td>
			<td>Tecan</td>
			<td>Infinite 200 PRO</td>
			<td>https://lifesciences.tecan.com/plate_readers/infinite_200_pro</td>
		</tr>
		<tr>
			<td>VWR Benchmark Incu-shaker 10L</td>
			<td>VWR</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray power defraction</td>
			<td>&#160;Panalytical</td>
			<td>N/A</td>
			<td>PANalytical Empyrean Series 2</td>
		</tr>
	</tbody>
</table>

evaluation of antimicrobial activities of nanoparticles and nanostructured surfaces in vitro

The antimicrobial activities of nanoparticles and nanostructured surfaces, such as silver, zinc oxide, titanium dioxide, and magnesium oxide, have been explored previously in clinical and environmental settings and in consumable food products. However, a lack of consistency in the experimental methods and materials used has culminated in conflicting results, even amongst studies of the same nanostructure types and bacterial species. For researchers who wish to employ nanostructures as an additive or coating in a product design, these conflicting data limit their utilization in clinical settings. 
To confront this dilemma, in this article, we present four different methods to determine the antimicrobial activities of nanoparticles and nanostructured surfaces, and discuss their applicability in different scenarios. Adapting consistent methods is expected to lead to reproducible data that can be compared across studies and implemented for different nanostructure types and microbial species. We introduce two methods to determine the antimicrobial activities of nanoparticles and two methods for the antimicrobial activities of nanostructured surfaces. 
For nanoparticles, the direct co-culture method can be used to determine the minimum inhibitory and minimum bactericidal concentrations of nanoparticles, and the direct exposure culture method can be used to assess real-time bacteriostatic versus bactericidal activity resulting from nanoparticle exposure. For nanostructured surfaces, the direct culture method is used to determine the viability of bacteria indirectly and directly in contact with nanostructured surfaces, and the focused-contact exposure method is used to examine antimicrobial activity on a specific area of a nanostructured surface. We discuss key experimental variables to consider for in vitro study design when determining the antimicrobial properties of nanoparticles and nanostructured surfaces. All these methods are relatively low cost, employ techniques that are relatively easy to master and repeatable for consistency, and are applicable to a broad range of nanostructure types and microbial species.

The identification of the antibacterial activity of magnesium oxide nanoparticles and nanostructured surfaces has been presented using four in vitro methods that are applicable across different material types and microbial species.
Method A and method B examine bacterial activities when exposed to nanoparticles at a lag phase (method A) and log phase (method B) for a duration of 24 h or longer. Method A provides results regarding the MIC and MBC, while method B determines the inhibito...

Watch this Scientific Journal Video about Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro at JoVE.com

Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro

We introduce four methods to evaluate the antimicrobial activities of nanoparticles and nanostructured surfaces using in vitro techniques. These methods can be adapted to study the interactions of different nanoparticles and nanostructured surfaces with a broad range of microbial species.

Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro

The antimicrobial activities of nanoparticles and nanostructured surfaces, such as silver, zinc oxide, titanium dioxide, and magnesium oxide, have been ...

The protocols provide a method to examine the antimicrobial activities of nanoparticle and nanostructured surfaces from preliminary stage investigations to more complex surface examinations. Unlike previously described methods that resulted in incomparable data across studies, these methods produce consistent and comparable results across different nanoparticle materials, nanostructured materials, and microbial species. Maintaining a homogenous distribution of nanoparticles in methods A and B can be difficult.Vortexing samples thoroughly will ensure nanoparticle suspension, and pipetting three to four times between aliquots will maintain suspensions Begin collecting cultured bacteria by aliquoting one milliliter of the overnight grown bacterial culture into 1.5 milliliter micro centrifuge tubes. After creating a sufficient number of aliquots, centrifuge them to reach the desired seating density. Collect the supernatant into a collection receptacle.And re-suspend the cell pellet in 0.5 milliliters of fresh growth medium. Combine the suspension from two tubes, and repeat the centrifugalization. After centrifugalization, give a second wash with fresh growth medium and repeat the combining of the content from two tubes into one before centrifuging the tubes.Give the third wash with 0.33 milliliters of tris buffer or hydroxyethyl aminomethane buffer. Combine the three suspensions, each of 0.33 milliliter volume, into one 1.5 milliliter micro centrifuge. After centrifugalization, remove the supernatant from the pelleted cells, and re-suspend the cell pellet in one milliliter of fresh tris buffer which will be known as the cell suspension or a lag phase bacterial seeding culture.To form the bacteria and nanoparticle cultures, aliquot the two milliliters of the bacterial seeding culture in each well of a 12 well non-tissue treated polystyrene plate. For each five milliliter micro centrifuge tube containing pre-weighed magnesium oxide particles, add three milliliters of the bacterial seeding culture. And vortex the tube briefly to mix the magnesium oxide nanoparticles with the bacteria.After mixing, aliquot one milliliter of the magnesium oxide nanoparticles suspension to three separate wells to create the triplicate samples of each premeasured weight of magnesium oxide nanoparticles. Incubate the sample overnight at 37 degrees Celsius and 120 rpm. And transfer a three milliliter sample into an individual 15 milliliter conical tube using a pipette.Then perform one to 10 serial dilution in a 96 well plate by adding 180 microliters of tris buffer to each well in row B to row G for the appropriate number of columns. Briefly vortex a 15 milliliter conical tube containing bacterial samples, and add 50 microliters of bacterial sample to an individual well in row A.Next, from row A, transfer 20 microliters of bacterial sample into the corresponding well of row B with brief mixing. Using a sterile pipette tip, transfer 20 microliters from the well in row B to the corresponding well in row C.Continue this until row G to complete the serial dilution from 10 to the negative one in row B to 10 to the negative six in row G.Once done, pipette 100 microliters of each well into an appropriate growth agar plate, and spread across the plate to disperse the cell culture.Place the well plates into an incubator shaker overnight at 37 degrees Celsius, and incubate the agar plates at 37 degrees Celsius for 24 hours. Examine the plate, and count the plates having approximately 25 to 300 colonies. If possible, choose plates of the same dilution value.For each material to be tested, dilute the overnight bacterial samples by adding three 200 microliter aliquots to the growth medium into three wells, and three 200 microliter aliquots of the bacterial culture into additional wells. Place the 96 well plate into the plate reader and scan it. If necessary, continue adding broth or overnight bacterial culture until the optical density at 600 nanometers, or OD 600, is reached approximately 0.01.To prepare bacterial nanoparticle suspensions and sterile media nanoparticle suspensions, remove one milliliter of bacterial culture or medium from a triplicate set of 15 milliliter conical tubes. And add it into a five milliliter centrifuge tube containing premeasured nanoparticles. Vortex the tubes to mix the solution.Distribute the nanoparticles homogeneously by transferring one milliliter aliquots from the five milliliter centrifuge tube to the 15 milliliter conical tube. Once done, aliquot 200 microliters of each sample into the individual wells of a 96 well plate. After determining the seating density described earlier, add the samples and control into the individual wells of a 48 well non-tissue treated polystyrene plate.Next, aliquot the 0.75 milliliters of the cell suspension into each well containing the samples and controls. Place the 48 well plate into an incubator shaker for 24 hours at 37 degrees Celsius, with shaking at 120 RPM. After incubation, collect two samples from each group, and transfer them individually into labeled five milliliter micro centrifuge tubes.Add two milliliters of revised simulated body fluid, or RSBF, to each tube. Prepare sterilized nitrous cellulose papers by trimming them into one centimeter of diameter. Place the cut nitrocellulose paper onto an agar plate containing the appropriate medium.Next, add 50 microliters of the diluted bacterial culture onto the filter paper before adding 50 microliters of an appropriate medium to the center of each sample surface. Using sterilized tweezers, pick up the nitric cellose paper from the surface of the auger. Carefully flip the nitric cellulose paper and place it onto the sample surface so that the bacteria are in contact with the 50 microliters of medium, and the nanostructured surface of interest.If the sample is degradable in culture, put a holder underneath it to lift it to avoid contact with the tris buffer. Maintain the humidity by adding one milliliter of tris buffer to a sample containing well. After overnight incubation, collect the nitrocellulose paper from each sample surface and place them into five milliliters of tris buffer.Vortex the collected filter papers and nanosurface material samples for five seconds. Collect the tris buffer suspensions from the sample and place them into individual fresh collection tubes. The antimicrobial effects using method A identified minimum and inhibitory concentration, or MIC, of one milligram per milliliter of magnesium oxide nanoparticles, and minimum bacteriocidal concentrations, or MBC 99.9, of 1.0 and 1.6 milligrams per milliliter of magnesium oxide nanoparticles for gram-negative Escherichia coli and pseudomonas aeruginosa respectively.The gram-positive staphylococcus epidermis, S.Aureus, and methicillin-resistant staphylococcus aureus, or MRSA, demonstrated the MIC's values of 0.5, 0.7, and one milligrams per milliliter magnesium oxide nanoparticles respectively. NBC 99.99 values of 1.6 and 1.2 milligrams per milliliter were identified for S.epidermis and S.aureus respectively. While MRSA was not reduced beyond NBC 90.MICs of 1.2 and one milligram per milliliter were identified in drug sensitive and drug-resistant candida species, C albicans, and C albicans fluconazole resistant, or FR respectively. In contrast, magnesium oxide nanoparticles demonstrated MIC values of one and 0.7 milligrams per milliliter, or C.glabrata and C glabrata echinocandin resistant, or ER respectively. Each candidate species reached an NBC 90 of 0.7 to 1.2 milligrams per milliliter, but only C.glabrata ER was reduced to NBC 99.9 at 1.2 milligrams per milliliter.In method B, MRSA exposed to nanoparticles grew exponentially to an OD of 0.85. Exposure to 1.2 milligrams per milliliter of magnesium oxide nanoparticles and 6.25 milligrams per milliliter of trimethoprim resulted in 80.2%and 81.6%of reduced bacterial growth respectively. Similarly, 2.9 milligrams per milliliter of magnesium hydroxide nanoparticles reduced bacterial growth to 70.3%Exposure to one microliter per milliliter of vancomycin resulted in a 99.99%reduction in the growth of MRSA, suggesting the bacteriostatic activity of magnesium oxide and magnesium hydroxide nanoparticles.Method C showed no inhibition of bacterial growth with indirect contact. The results indicated that ZC21 had the strongest antibacterial activity against the adhesion and growth of MRSA, for all the samples tested. In Method D, no viable staph aureus was identified in the 1.9A, 1.9 AA, and EPD samples, or their paired filter papers.However, exposure to the EPD samples after a kneeling reduced bacterial growth to a few cells on the nanostructured surface and the pared filter paper. Identifying nanoparticle and nanostructured surface antimicrobial activities has led to additional investigations in bioengineering applications, including those related to chronic wounds and implant-related infections.

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JoVE Journal

Bioengineering

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...