This work was funded by the United States Department of Defense through the Congressionally Directed Medical Research Programs (CDMRP) under contract W81XWH-17-1-0085, and the National Institute of Health R03 EB025903-1. AV was funded by the College of Engineering Dean&#8217;s Fellowship at Northeastern University.

University approvals were obtained for conducting the experiments with dead tissues. Bovine joints were obtained commercially from a slaughterhouse.
1. Cartilage explant extraction
<ol>
	<li>Using a scalpel (#10 blade), cut and remove fat, muscles, ligaments, tendons and all other connective tissue to expose the cartilage from the femoropatellar groove of bovine knee joints.</li>
	<li>Using 3 mm and 6 mm dermal punches, make perpendicular punches into the cartilage to extract cylindrical plugs. Immediately place the plugs in individual wells of a 48-well plate containing 500 &#956;L of 1x phosphate buffered saline (PBS) supplemented with 1% v/v antibiotic-antimycotic.</li>
	<li>Place the superficial side of a cartilage plug facing down into a well in the slicing fixture (Figure 2). Using a razor blade, slice the plug along the surface of the slicing fixture to obtain a 1 mm thick cartilage explant that is inclusive of the superficial zone. Repeat for each cartilage plug.</li>
	<li>Store cartilage explants individually in polypropylene tubes containing 500 &#956;L of 1x PBS supplemented with protease inhibitors (PBS-PI, 1 PI mini-tablet per 50 mL 1x PBS) at -20 &#176;C.</li>
	<li>Prior to conducting each of the following transport experiments, thaw the explant-containing vials for 30 min in a 37 &#176;C water bath.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61340/61340fig02.jpg" /> 
Figure 2: Custom-designed slicing fixture.&#160;Design parameters of stainless steel slicing fixture used for slicing cartilage explants of 3 and 6 mm diameter. Plastic inserts of varying thickness were placed inside wells to adjust the thickness of sliced explants. Stainless steel cylindrical pin of &#60;1 mm diameter was used to push explant out of fixture. All numerical values are presented in mm. <a href="https://www.jove.com/files/ftp_upload/61340/61340fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Equilibrium uptake of CPCs in cartilage
<ol>
	<li>Gently dab cartilage explants (3 mm dia. X 1 mm thick.) with a delicate task wipe to remove excess 1x PBS from the explant surface. Using a balance, quickly record the wet weight of each explant and then immediately place in a 1x PBS bath to prevent dehydration.</li>
	<li>Prepare 30 &#956;M solutions (300 &#956;L per explant) of fluorescently labeled CPCs in 1x PBS-PI. Use RNase-free polypropylene tubes for reconstitution.</li>
	<li>In a 96-well plate, pipette 300 &#956;L of each 30 &#956;M CPC solution into separate wells. Avoid using wells near the edge of the plate to prevent evaporation. Using a spatula, transfer each explant to the solution containing wells.</li>
	<li>Fill surrounding wells with 300 &#956;L of 1x PBS and cover the well plate with lid. Seal the edges of the plate with flexible film to minimize evaporation.</li>
	<li>Inside of a 37 &#176;C incubator, place the plate on a plate shaker to limit the particle sedimentation. Incubate for 24 h under gentle rotation (50 rpm with a 15 mm orbit) to allow for the equilibrium uptake of CPCs in the cartilage (Figure 3).</li>
	<li>Generate a standard curve for correlation of fluorescence to CPC concentration
	<ol>
		<li>Prepare serial dilutions of CPC solutions from 30 &#956;M &#8211; 0 &#956;M (10 2-fold dilutions) in 1x PBS-PI in polypropylene tubes. Ensure that at least 500 &#956;L of each dilution is present.</li>
		<li>Add 200 &#956;L of each dilution to consecutive wells in a black 96-well plate. Duplicate in another row to increase sample size.</li>
		<li>Obtain fluorescence readings of each sample using a plate reader at the excitation and emission wavelengths of the fluorescent label using a plate reader.</li>
		<li>Plot fluorescence reading vs. CPC concentration and derive an equation for the linear portion of the curve. 
		NOTE: To limit the variability in fluorescence readings, incubate the CPC stock solution under the same conditions as the sample plate prior to generation of the standard curve.</li>
	</ol>
	</li>
	<li>After 24 h of incubation, collect the equilibrium bath from each well in separate polypropylene tubes.</li>
	<li>Transfer 200 &#956;L of each solution into separate wells of a black 96-well plate. Obtain fluorescence readings of each sample under the same fluorescent settings as for the standard curve. If necessary, dilute the sample in 1x PBS-PI to ensure readings fall within the linear portion of the standard curve.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61340/61340fig03.jpg" /> 
Figure 3: Schematic of equilibrium uptake experiments.&#160;Cartilage explants (3 mm dia. x 1 mm thick) were placed in individuals wells in a 96-well plate containing fluorescently tagged CPC solution. After 24 h CPCs were uptaken by the cartilage, thereby reducing the fluorescence of the surrounding bath. <a href="https://www.jove.com/files/ftp_upload/61340/61340fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Depth of penetration of CPCs in the cartilage
<ol>
	<li>Prepare 30 &#956;M solutions (300 &#956;L per explant) of fluorescently labeled CPCs in 1x PBS-PI. Use RNase-free polypropylene tubes for reconstitution.</li>
	<li>Using a scalpel, cut cartilage explants (6 mm diameter x 1 mm thickness) in half to make half-disks. Keep the explant hydrated with a layer of 1x PBS-PI while cutting.</li>
	<li>Glue a half-disk explant into the middle of one well of the custom-designed 1-dimensional transport chamber using an epoxy (Figure 4, Figure 5). Ensure epoxy is applied to the circumferential (curved) side of the explant. Remove excess glue from the well to prevent contact with the diffusion surface area of cartilage and make a note of the superficial side of the explant.</li>
	<li>Add 80 &#956;L of 1x PBS-PI to both sides of the explant. Pipette the liquid up and down from one side of the explant to check for leakage to the other side. If leakage occurs, readjust explant and apply epoxy as needed.</li>
	<li>Replace the 1x PBS-PI from the side facing the superficial surface of cartilage (upstream) with 80 &#956;L of 30 &#956;M CPC solution. Maintain 80 &#956;L of 1x PBS-PI on the side facing the deep zone of cartilage (downstream).</li>
	<li>Carefully place the transport chamber in a coverable container. Cover the base of the container with a layer 1x PBS to avoid evaporation of solutions. Ensure that there is no direct contact between solutions from upstream and downstream chambers.</li>
	<li>Place the covered container&#160;on a plate shaker to limit particle sedimentation. Incubate for either 4 or 24 h at room temperature under gentle rotation (50 rpm with a 15 mm orbit).</li>
	<li>After incubation, remove the explant from chamber and cut ~100 &#956;m slice from the center of the explant. 
	NOTE: This cross-section is inclusive of the superficial, middle and deep zones of cartilage.</li>
	<li>Place the slice between a glass slide and a coverslip. Hydrate the slice with a layer of 1x PBS-PI.</li>
	<li>At 10x magnification, image through the full thickness of the slice to obtain z-stack of fluorescent images using a confocal microscope.</li>
	<li>Using ImageJ project the average intensity of the images within the z-stack to determine the depth of penetration of CPCs in cartilage.
	<ol>
		<li>Open the image stack by clicking on File | Open.</li>
		<li>Click on &#8216;Image&#8217; on the task bar and click Image | Stacks | Z Project from the dropdown menu.</li>
		<li>Input slice numbers from 1 to the final slice. Select &#8216;Average Intensity&#8217; under Projection Type. Click &#8216;OK.&#8217;</li>
	</ol>
	</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61340/61340fig04.jpg" /> 
Figure 4: Custom-designed 1-D transport chamber.&#160;Design parameters of PMMA 1D transport chamber with 6 individual wells. All numerical values are presented in mm. <a href="https://www.jove.com/files/ftp_upload/61340/61340fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61340/61340fig05.jpg" /> 
Figure 5: Schematic of depth of penetration studies.&#160;Cartilage explants (6 mm diameter x 1 mm thickness) were cut in half and fixed to the center of 1-D diffusive transport wells. Fluorescently tagged CPC solution was added to the side of the well in contact with the superficial zone (SZ) of cartilage. 1x PBS-PI was added to the side of the well in contact with the deep zone (DZ) of cartilage. Following diffusion, a cross-section of cartilage (3 mm x 1 mm) was imaged using confocal microscopy. This figure has been modified from Vedadghavami et al.4 and Bajpayee et al.3 <a href="https://www.jove.com/files/ftp_upload/61340/61340fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Non-equilibrium diffusion rate of CPCs in the cartilage
<ol>
	<li>Bring the two halves of the custom-designed transport chamber (Figure 6) together to assemble and close the chamber. Use washers, nuts and bolts to tightly close the chamber with a wrench. 
	NOTE: The transport chamber must be translucent as to not interfere with fluorescent readings. The transport chambers used in this protocol are made from polymethylmethacrylate (PMMA).</li>
	<li>Coat the inner space of the chamber with 0.5% w/v non-fat bovine milk solution in 1x PBS (2 mL for each chamber) for 15 min to prevent non-specific binding of CPCs to chamber walls. Then rinse the chamber with 1x PBS (2 mL for each chamber).</li>
	<li>Using the custom-designed slicing fixture (Figure 2) and a razor blade, slice a 6 mm diameter cartilage explant (transverse plane) to a thickness of 500-800 &#956;m, inclusive of the superficial zone. Keep the explant hydrated with 1x PBS.</li>
	<li>Using hammer-driven and dermal punches, create gaskets from rubber sheets as shown in Figure 7.</li>
	<li>Assemble each half transport chamber to include 1 large rubber gasket, 1 PMMA insert and 1 small rubber gasket each. Place the explant in the wells of the plastic insert, with the superficial zone facing the upstream chamber. Sandwich the two halves together to complete the assembly and screw tightly using a wrench (Figure 7).</li>
	<li>Fill the upstream chamber with 2 mL of 1x PBS-PI and observe the downstream chamber for leakage of fluid from the upstream chamber. If leakage is present, reassemble the chamber, adjusting gasket position and tightness of screws. If no leakage, fill the downstream chamber with 2 mL 1x PBS-PI as well.</li>
	<li>Add a mini-stir bar to both up and downstream chambers and place the chamber on a stir plate. Align the chamber so that laser from the spectrophotometer is focused towards the center of the downstream chamber. Place the signal receiver portion of the spectrophotometer behind the downstream chamber (Figure 8). 
	NOTE: The laser and receiver of the spectrophotometer must be equipped with the appropriate filters to excite, emit and transmit signals from the fluorescently labeled protein. Protect the transport chamber from light using a black box during experimentation to avoid interference in fluorescence signal. It is best practice to seal the openings on top of the chamber with flexible film to avoid evaporation.</li>
	<li>Collect real-time downstream fluorescence emission readings and ensure a stable signal for at least 5 min. 
	NOTE: Aliquots from the downstream chamber can be obtained and assessed for fluorescence using a plate reader if a custom-designed spectrophotometer or translucent transport chamber is not available.</li>
	<li>Pipette a pre-calculated volume of stock solution of fluorescently tagged CPCs into the upstream chamber to ensure a final concentration of 3 &#956;M inside the upstream chamber. Observe the downstream fluorescence signal and allow for solute transport to reach a steady increase in slope. 
	NOTE: A thicker cartilage explant will require longer time to reach steady-state.</li>
	<li>Once the steady state has been reached, take 20 &#956;L from the upstream chamber and add to the downstream chamber (&#8220;spike test&#8221;). 
	NOTE: A spike in the downstream fluorescence will be observed. This will allow for correlation between fluorescence readings and CPC concentration.</li>
	<li>Collect real-time downstream fluorescence readings.</li>
</ol>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61340/61340fig06.jpg" /> 
Figure 6: Custom-designed non-equilibrium diffusion transport chamber.&#160;Design parameters of PMMA non-equilibrium diffusion transport chamber. The chamber must be translucent as to not interfere with fluorescence readings. The complete transport chamber consisted of two identical halves of the fixture shown. Two cylindrical stainless-steel pins (~2.94 mm diameter, ~18 mm long) were required to ensure alignment and complete closure of the halves of the chamber. Four identical slots for 6-32 thread screws were made in each corner of the chamber for screw tight assembly. All numerical values are presented in millimeters. <a href="https://www.jove.com/files/ftp_upload/61340/61340fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61340/61340fig07.jpg" /> 
Figure 7: Assembly of non-equilibrium diffusion transport chamber.&#160;Design parameters of (A) black PMMA inserts and (B) large and small rubber gaskets. Thickness of rubber gaskets was adjusted to ensure tight closure of the chamber. All numerical values are presented in mm. (C) Schematic showing the order of assembly for two halves of transport chamber with cartilage explant placed in the center. SZ indicates superficial zone of cartilage which was facing the upstream chamber. <a href="https://www.jove.com/files/ftp_upload/61340/61340fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61340/61340fig08.jpg" /> 
Figure 8: Schematic of non-equilibrium diffusion experiments.&#160;Cartilage explants (6 mm diameter x 1 mm thickness) were placed in the center of the transport chamber with the superficial surface facing the upstream chamber. Both up and downstream sides of the chamber were filled with 1x PBS-PI and mixed using a mini stir bar. With a laser pointed towards the downstream chamber to collect fluorescent readings, fluorescently tagged CPC solution was added to the upstream chamber. <a href="https://www.jove.com/files/ftp_upload/61340/61340fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+of+articular+cartilage+thickness+in+the+stifle+of+animal+species+used+in+human+pre-clinical+studies+compared+to+articular+cartilage+thickness+in+the+human+knee.">A comparative study of articular cartilage thickness in the stifle of animal species used in human pre-clinical studies compared to articular cartilage thickness in the human knee.</a> Veterinary and Comparative Orthopaedics and Traumatology. 19 (3), 142-146 (2006).</li></ol>

The authors have nothing to disclose.

The methods and protocols described here are significant to the field of targeted drug delivery to negatively charged tissues. Due to the high density of negatively charged aggrecans present in these tissues, a barrier is created, thus preventing drugs from reaching their cellular target sites which lie deep within the matrix. To address this outstanding challenge, drugs can be modified to incorporate positively charged drug carriers which can enhance the transport rate, uptake and binding of drugs within tissue<sup clas...

Drug-delivery to negatively charged tissues in the body remains a challenge due to the inability of drugs to penetrate deep into the tissue to reach cell and matrix target sites1. Several of these tissues comprise of densely-packed, negatively-charged aggrecans which create a high negative fixed charge density (FCD)2 within the tissue and act as a barrier for the delivery of most macromolecules3,4. However, with the assistance of positively charged drug carriers, this negatively charged tissue barrier can actually be converted into a drug depot via electrostatic charge interactions for sustained drug delivery1,5,6,7(Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61340/61340fig01.jpg" /> 
Figure 1: Charge based intra-cartilage delivery of CPCs.&#160;Intra-articular injection of CPCs into the knee joint space. Electrostatic interactions between positively charged CPCs and negatively charged aggrecan groups enable rapid and full depth penetration through cartilage. This figure has been modified from Vedadghavami et al4. <a href="https://www.jove.com/files/ftp_upload/61340/61340fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Recently, short-length cationic peptide carriers (CPCs) were designed with the goal of creating small cationic domains capable of carrying larger sized therapeutics for delivery to the negatively charged cartilage4. For effective drug delivery to the cartilage for treating prevalent8,9 and degenerative diseases such as osteoarthritis (OA)10, it is critical that therapeutic concentrations of drugs penetrate deep within the tissue, where a majority of the cartilage cells (chondrocytes) lie11. Although there are several potential disease modifying drugs available, none have gained FDA approval because these are unable to effectively target the cartilage12,13. Therefore, evaluation of the transport properties of drug carriers is necessary for predicting the effectiveness of drugs in inducing a therapeutic response. Here, we have designed three separate experiments that can be utilized for assessing the equilibrium uptake, depth of penetration and non-equilibrium diffusion rate of CPCs4.
To ensure that there is a sufficient drug concentration within the cartilage that can provide an optimal therapeutic response, uptake experiments were designed to quantify equilibrium CPC concentration in cartilage4. In this design, following an equilibrium between the cartilage and its surrounding bath, the total amount of solute inside the cartilage (either bound to the matrix or free) can be determined using an uptake ratio. This ratio is calculated by normalizing the concentration of solutes inside the cartilage to that of the equilibrium bath. In principle, neutral solutes, whose diffusion through the cartilage is not assisted by charge interactions, would have an uptake ratio of less than 1. Conversely, cationic solutes, whose transport is enhanced via electrostatic interactions, show an uptake ratio greater than 1. However, as shown with CPCs, use of an optimal positive charge can result in much higher uptake ratios (greater than 300)4.
Although high drug concentration within the cartilage is important for achieving therapeutic benefit, it is also critical that drugs diffuse through the full thickness of the cartilage. Therefore, studies showing the depth of penetration are required to ensure that drugs reach deep within the cartilage so that the matrix and cellular target sites can be reached, thereby providing a more effective therapy. This experiment was designed to assess the one-way diffusion of solutes through cartilage, simulating diffusion of drugs into cartilage following intra-articular injection in vivo. Fluorescence imaging using confocal microscopy allows for the evaluation of depth of penetration into cartilage. Net particle charge plays a key role in moderating how deep drugs can diffuse through the matrix. An optimal net charge based on a tissue FCD is required to allow for weak-reversible binding interactions between cationic particles and the anionic tissue matrix. This implies that any interaction is weak enough so that particles can disassociate from the matrix but reversible in nature so that it can bind to another matrix binding site deeper within the tissue4. Conversely, excessive positive net charge of a particle can be detrimental towards diffusion, as too strong matrix binding prevents detachment of particles from the initial binding site in the superficial zone of cartilage. This would result in an insufficient biological response as a majority of the target sites lie deep within the tissue11.
To further quantify the strength of the binding interactions, analysis of drug diffusion rates through cartilage is advantageous. Non-equilibrium diffusion studies allow for the comparison of real-time diffusion rates between different solutes. As drugs diffuse through the superficial, middle and deep zones of cartilage, the presence of binding interactions can greatly alter diffusion rates. When binding interactions are present between drugs and the cartilage matrix, it is defined as the effective diffusivity (DEFF). In this case, once all binding sites have been occupied, the diffusion rate of drugs is governed by the steady-state diffusion (DSS). Comparison between the DEFF of different solute determines the relative binding strength of solutes with the matrix. For a given solute, if the DEFF and DSS are within the same order of magnitude, it implies that there is minimal binding present between the drug and matrix during diffusion. However, if DEFF is greater than DSS, substantial binding of particles to matrix exists.
The designed experiments individually allow for the characterization of solute transport through the cartilage, however, a holistic analysis inclusive of all results is required for designing an optimally charged drug carrier. The weak and reversible nature of charge interactions controls particle diffusion rate and allows for high equilibrium uptake and rapid full depth penetration through cartilage. Through equilibrium uptake experiments, we should look for carriers that show high uptake as a result of charge interactions which can be verified using non-equilibrium diffusion rate studies. However, these binding interactions should be weak and reversible in nature to allow for full-thickness penetration of the solute through cartilage. An ideal drug carrier would possess an optimal charge which enables strong enough binding for uptake and high intra-cartilage drug concentrations, but not too strong as to impede full-thickness diffusion4. The presented experiments will assist in the design characteristics for charge-based tissue targeting drug carriers. These protocols were used for characterizing CPC transport through cartilage4, however, these can also be applied to a variety of drugs and drug carriers through cartilage and other negatively charged tissues.

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			<td>316 Stainless Steel SAE Washer</td>
			<td>McMaster-Carr</td>
			<td>91950A044</td>
			<td>For number 5 screw size, 0.14&#34; ID, 0.312&#34; OD</td>
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		<tr>
			<td>96-Well Polystyrene Plate</td>
			<td>Fisherbrand</td>
			<td>12566620</td>
			<td>Black</td>
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		<tr>
			<td>Acrylic Thick Gauge Sheet</td>
			<td>Reynolds Polymer</td>
			<td>N/A</td>
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		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td>100x</td>
		</tr>
		<tr>
			<td>Bovine Cartilage</td>
			<td>Research 87</td>
			<td>N/A</td>
			<td>2-3 weeks old, femoropatellar groove</td>
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		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher BioReagents</td>
			<td>BP671-1</td>
			<td></td>
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		<tr>
			<td>CPC+14</td>
			<td>LifeTein</td>
			<td>LT1524</td>
			<td>Custom designed peptide</td>
		</tr>
		<tr>
			<td>CPC+20</td>
			<td>LifeTein</td>
			<td>LT1525</td>
			<td>Custom designed peptide</td>
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		<tr>
			<td>CPC+8</td>
			<td>LifeTein</td>
			<td>LT1523</td>
			<td>Custom designed peptide</td>
		</tr>
		<tr>
			<td>Delicate Task Wipers</td>
			<td>Kimberly-Clark Professional</td>
			<td>34155</td>
			<td></td>
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		<tr>
			<td>Dermal Punch</td>
			<td>MedBlades</td>
			<td>MB5-1</td>
			<td>3, 4 and 6 mm</td>
		</tr>
		<tr>
			<td>Economy Plain Glass Microscope Slides</td>
			<td>Fisherbrand</td>
			<td>12550A3</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat Bottom Cell Culture Plates</td>
			<td>Corning Costar</td>
			<td>3595</td>
			<td>Clear, 96 well</td>
		</tr>
		<tr>
			<td>Flexible Wrapping Film</td>
			<td>Bemis Parafilm M Laboratory</td>
			<td>1337412</td>
			<td></td>
		</tr>
		<tr>
			<td>Gold Seal Cover Glass</td>
			<td>Electron Microscopy Sciences</td>
			<td>6378701</td>
			<td># 1.5, 18x18 mm</td>
		</tr>
		<tr>
			<td>Hammer-Driven Hole Punch</td>
			<td>McMaster-Carr</td>
			<td>3427A15</td>
			<td>1/2&#34; Diameter</td>
		</tr>
		<tr>
			<td>Hammer-Driven Hole Punch</td>
			<td>McMaster-Carr</td>
			<td>3427A19</td>
			<td>3/4&#34; Diameter</td>
		</tr>
		<tr>
			<td>Laser</td>
			<td>Chroma Technology</td>
			<td>AT480/30m</td>
			<td>Spectrophotometer Laser Light</td>
		</tr>
		<tr>
			<td>Low-Strength Steel Hex Nut</td>
			<td>McMaster-Carr</td>
			<td>90480A007</td>
			<td>6-32 Thread size</td>
		</tr>
		<tr>
			<td>LSM 700 Confocal Microscope</td>
			<td>Zeiss</td>
			<td>LSM 700</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Magnetic Stirring Bars</td>
			<td>Bel-Art Spinbar</td>
			<td>F37119-0007</td>
			<td>7x2 mm</td>
		</tr>
		<tr>
			<td>Multipurpose Neoprene Rubber Sheet</td>
			<td>McMaster-Carr</td>
			<td>1370N12</td>
			<td>1/32&#34; Thickness</td>
		</tr>
		<tr>
			<td>Non-Fat Dried Bovine Milk</td>
			<td>Sigma Aldrich</td>
			<td>M7409</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish</td>
			<td>Chemglass Life Sciences</td>
			<td>CGN1802145</td>
			<td>150 mm diameter</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline</td>
			<td>Corning</td>
			<td>21-040-CMR</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Plate Shaker</td>
			<td>VWR</td>
			<td>89032-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitors</td>
			<td>Thermo Scientific</td>
			<td>A32953</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor Blades</td>
			<td>Fisherbrand</td>
			<td>12640</td>
			<td></td>
		</tr>
		<tr>
			<td>R-Cast Acrylic Thin Gauge Sheet</td>
			<td>Reynolds Polymer</td>
			<td>N/A</td>
			<td>Black transport chamber inserts</td>
		</tr>
		<tr>
			<td>RTV Silicone</td>
			<td>Loctite</td>
			<td>234323</td>
			<td>Epoxy, Non-corrosive, clear</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>TedPella</td>
			<td>549-3</td>
			<td>#10, #11 blades</td>
		</tr>
		<tr>
			<td>Signal Receiver</td>
			<td>Chroma Technology</td>
			<td>ET515lp</td>
			<td>Spectrophotometer Laser Signal Receiver</td>
		</tr>
		<tr>
			<td>Snap-Cap Microcentrifuge Tubes</td>
			<td>Eppendorf</td>
			<td>22363204</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Spatula</td>
			<td>TedPella</td>
			<td>13508</td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy H1 Microplate Reader</td>
			<td>Biotek</td>
			<td>H1M</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc-Plated Alloy Steel Socket Head Screw</td>
			<td>McMaster-Carr</td>
			<td>90128A153</td>
			<td>6-32 Thread size, 1&#34; Long</td>
		</tr>
	</tbody>
</table>

characterization of intra-cartilage transport properties of cationic peptide carriers

Several negatively charged tissues in the body, like cartilage, present a barrier to the targeted drug delivery due to their high density of negatively charged aggrecans and, therefore, require improved targeting methods to increase their therapeutic response. Because cartilage has a high negative fixed charge density, drugs can be modified with positively charged drug carriers to take advantage of electrostatic interactions, allowing for enhanced intra-cartilage drug transport. Studying the transport of drug carriers is, therefore, crucial towards predicting the efficacy of drugs in inducing a biological response. We show the design of three experiments which can quantify the equilibrium uptake, depth of penetration and non-equilibrium diffusion rate of cationic peptide carriers in cartilage explants. Equilibrium uptake experiments provide a measure of the solute concentration within the cartilage compared to its surrounding bath, which is useful for predicting the potential of a drug carrier in enhancing therapeutic concentration of drugs in cartilage. Depth of penetration studies using confocal microscopy allow for the visual representation of 1D solute diffusion from the superficial to deep zone of cartilage, which is important for assessing whether solutes reach their matrix and cellular target sites. Non-equilibrium diffusion rate studies using a custom-designed transport chamber enables the measurement of the strength of binding interactions with the tissue matrix by characterizing the diffusion rates of fluorescently labeled solutes across the tissue; this is beneficial for designing carriers of optimal binding strength with cartilage. Together, the results obtained from the three transport experiments provide a guideline for designing optimally charged drug carriers which take advantage of weak and reversible charge interactions for drug delivery applications. These experimental methods can also be applied to evaluate the transport of drugs and drug-drug carrier conjugates. Further, these methods can be adapted for the use in targeting other negatively charged tissues such as meniscus, cornea and the vitreous humor.

Following equilibrium absorption of CPCs by cartilage, the bath fluorescence decreases when the solute has been uptaken by the tissue. However, if the fluorescence value of the final bath remains similar to the initial, it indicates that there is no/minimal solute uptake. Another confirmation of solute uptake is if the tissue has visibly changed color to the color of the fluorescent dye. The quantitative uptake of solutes in cartilage was determined using the uptake ratio (RU) after the fluorescence values wer...

Watch this Scientific Journal Video about Characterization of Intra-Cartilage Transport Properties of Cationic Peptide Carriers at JoVE.com

Characterization of Intra-Cartilage Transport Properties of Cationic Peptide Carriers

This protocol determines equilibrium uptake, depth of penetration and non-equilibrium diffusion rate for cationic peptide carriers in cartilage. Characterization of transport properties is critical for ensuring an effective biological response. These methods can be applied for designing an optimally charged drug carriers for targeting negatively charged tissues.

Several negatively charged tissues in the body, like cartilage, present a barrier to the targeted drug delivery due to their high density of negatively ...

characterization-intra-cartilage-transport-properties-cationic

Research

JoVE Journal

Bioengineering

7.4K Views.  Northeastern University. This protocol determines equilibrium uptake, depth of penetration and non-equilibrium diffusion rate for cationic peptide carriers in cartilage. Characterization of transport properties is critical for ensuring an effective biological response. These methods can be applied for designing an optimally charged drug carriers for targeting negatively charged tissues.

Video: Characterization of Intra-Cartilage Transport Properties of Cationic Peptide Carriers

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson&#8217;s disease and beta-amyloid and tau in Alzheimer&#8217;s disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases.

Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis.

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Transport Properties of Ibuprofen Encapsulated in Cyclodextrin Nanosponge Hydrogels: A Proton HR-MAS NMR Spectroscopy Study

The chemical cross-linking of &#946;-cyclodextrin (&#946;-CD) with ethylenediaminetetraacetic dianhydride (EDTA) led to branched polymers referred to as cyclodextrin nanosponges (CDNSEDTA). Two different preparations are described with 1:4 and 1:8 CD-EDTA molar ratios. The corresponding cross-linked polymers were contacted with 0.27 M aqueous solution of ibuprofen sodium salt (IP) leading to homogeneous, colorless, drug loaded hydrogels.
The systems were characterized by high resolution magic angle spinning (HR-MAS) NMR spectroscopy. Pulsed field gradient spin echo (PGSE) NMR spectroscopy was used to determine the mean square displacement (MSD) of IP inside the polymeric gel at different observation times td. The data were further processed in order to study the time dependence of MSD: MSD = f(td). The proposed methodology is useful to characterize the different diffusion regimes that, in principle, the solute may experience inside the hydrogel, namely normal or anomalous diffusion. The full protocols including the polymer preparation and purification, the obtainment of drug-loaded hydrogels, the NMR sample preparation, the measurement of MSD by HR-MAS NMR spectroscopy and the final data processing to achieve the time dependence of MSD are here reported and discussed. The presented experiments represent a paradigmatic case and the data are discussed in terms of innovative approach to the characterization of the transport properties of an encapsulated guest within a polymeric host of potential application for drug delivery.

The motion regimes of ibuprofen encapsulated in &#946;-cyclodextrin nanosponges polymer network are investigated using pulsed-field-gradient spin-echo (PGSE) NMR technique. Synthesis, purification, drug loading, implementation of the NMR pulse sequence and data analysis to work out the mean square displacement of the drug at several observation times are described in detail.

The chemical cross-linking of &#946;-cyclodextrin (&#946;-CD) with ethylenediaminetetraacetic dianhydride (EDTA) led to branched polymers referred to as ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. Atherosclerosis is also the leading cause of sudden death and myocardial infarction, instigated by unstable plaques that rupture and occlude the blood vessel without warning. Current imaging modalities cannot differentiate between stable and unstable plaques that rupture. Peptide amphiphiles micelles (PAMs) can overcome this drawback as they can be modified with a variety of targeting moieties that bind specifically to diseased tissue. Monocytes have been shown to be early markers of atherosclerosis, while large accumulation of monocytes is associated with plaques prone to rupture. Hence, nanoparticles that can target monocytes can be used to discriminate different stages of atherosclerosis. To that end, here, we describe a protocol for the preparation of monocyte-targeting PAMs (monocyte chemoattractant protein-1 (MCP-1) PAMs). MCP-1 PAMs are self-assembled through synthesis under mild conditions to form nanoparticles of 15 nm in diameter with near neutral surface charge. In vitro, PAMs were found to be biocompatible and had a high binding affinity for monocytes. The methods described herein show promise for a wide range of applications in atherosclerosis as well as other inflammatory diseases.

This paper presents a method involving the synthesis and characterization of monocyte-targeting peptide amphiphile micelles and the corresponding assays to test for biocompatibility and ability of the micelle to bind to monocytes.

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. ...

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive properties of in vitro transcribed (IVT) mRNA. With the help of IVT mRNA, the de novo synthesis of a desired protein can be induced without changing the physiological state of the target cell. Moreover, protein biosynthesis can be precisely controlled due to the transient effect of IVT mRNA.
For the efficient transfection of cells, nanoliposomes (NLps) may represent a safe and efficient delivery vehicle for therapeutic mRNA. This study describes a protocol to generate safe and efficient cationic NLps consisting of DC-cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a delivery vector for IVT mRNA. NLps having a defined size, a homogeneous distribution, and a high complexation capacity, and can be produced using the dry-film method. Moreover, we present different test systems to analyze their complexation and transfection efficacies using synthetic enhanced green fluorescent protein (eGFP) mRNA, as well as their effect on cell viability. Overall, the presented protocol provides an effective and safe approach for mRNA complexation, which may advance and improve the administration of therapeutic mRNA.

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA.

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...