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.

<table><tbody><tr><td>316 Stainless Steel SAE Washer</td><td>McMaster-Carr</td><td>91950A044</td><td>For number 5 screw size, 0.14&quot; ID, 0.312&quot; OD</td></tr><tr><td>96-Well Polystyrene Plate</td><td>Fisherbrand</td><td>12566620</td><td>Black</td></tr><tr><td>Acrylic Thick Gauge Sheet</td><td>Reynolds Polymer</td><td>N/A</td><td>For non-equilibrium diffusion and 1-D diffusion transport chamber</td></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></tr><tr><td>Bovine Serum Albumin</td><td>Fisher BioReagents</td><td>BP671-1</td><td></td></tr><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></tr><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></tr><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&quot; Diameter</td></tr><tr><td>Hammer-Driven Hole Punch</td><td>McMaster-Carr</td><td>3427A19</td><td>3/4&quot; 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&quot; 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&quot; 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

7.5K Views.  Northeastern University. Het karakteriseren van de transporteigenschappen van therapieën en hun dragers is van cruciaal belang voor het waarborgen van effectieve biologische reacties. Deze methoden helpen bij het ontwerpen van optimaal geladen geneesmiddelendragers voor het richten van negatief geladen weefsels. Het belangrijkste voordeel van deze technieken is hun vermogen om in vivo therapeutische werkzaamheid beter te voorspellen door middel van een reeks in vitro experimenten die solute transport door weefsel ka...

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

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

Biochemistry

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

Genetics

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

Synthesis and Characterization of Placental Chondroitin Sulfate A (plCSA)-Targeting Lipid-Polymer Nanoparticles

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising approach to cancer therapy. The glycosaminoglycan placental chondroitin sulfate A (plCSA) is expressed on a wide range of cancer cells and placental trophoblasts, and malarial protein VAR2CSA can specifically bind to plCSA. A reported placental chondroitin sulfate A binding peptide (plCSA-BP), derived from malarial protein VAR2CSA, can also specifically bind to plCSA on cancer cells and placental trophoblasts. Hence, plCSA-BP-conjugated nanoparticles could be used as a tool for targeted drug delivery to human cancers and placental trophoblasts. In this protocol, we describe a method to synthesize plCSA-BP-conjugated lipid-polymer nanoparticles loaded with doxorubicin (plCSA-DNPs); the method consists of a single sonication step and bioconjugate techniques. In addition, several methods for characterizing plCSA-DNPs, including determining their physicochemical properties and cellular uptake by placental choriocarcinoma (JEG3) cells, are described.

Synthesis and Characterization of Placental Chondroitin Sulfate A plCSA-Targeting Lipid-Polymer Nanoparticles

Here, we present a protocol for the synthesis of placental chondroitin sulfate A binding peptide (plCSA-BP)-conjugated lipid-polymer nanoparticles via single-step sonication and bioconjugate techniques. These particles constitute a novel tool for the targeted delivery of therapeutics to most human tumors and placental trophoblasts to treat cancers and placental disorders.

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising ...

Cancer Research

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

Assembly and Characterization of Polyelectrolyte Complex Micelles

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful platform for exploring the physics of polyelectrolyte interactions and also offer a promising solution to the pressing problem of delivering therapeutic oligonucleotides in vivo. Developing predictive structure-property relationships for PCMs has proven difficult, in part due to the presence of strong kinetic traps during nanoparticle self-assembly. This article discusses criteria for choosing polymers for PCM construction and provides protocols based on salt annealing that enable assembly of repeatable, low-polydispersity nanoparticles. We also discuss PCM characterization using light scattering, small-angle X-ray scattering, and electron microscopy.

We provide protocols and representative data for designing, assembling, and characterizing polyelectrolyte complex micelles, core-shell nanoparticles formed by polyelectrolytes and hydrophilic charged-uncharged block copolymers.

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful ...

Chemistry

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular cartilage impairs its load-bearing function substantially as it experiences tremendous chemical degradation, i.e. proteoglycan loss and collagen fibril disruption. One promising way to investigate chemical damage mechanisms during OA is to expose the cartilage specimens to an external solute and monitor the diffusion of the molecules. The degree of cartilage damage (i.e. concentration and configuration of essential macromolecules) is associated with collisional energy loss of external solutes while moving across articular cartilage creates different diffusion characteristics compared to healthy cartilage. In this study, we introduce a protocol, which consists of several steps and is based on previously developed experimental micro-Computed Tomography (micro-CT) and finite element modeling. The transport of charged and uncharged iodinated molecules is first recorded using micro-CT, which is followed by applying biphasic-solute and multiphasic finite element models to obtain diffusion coefficients and fixed charge densities across cartilage zones.

We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods.

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

Construction of Cyclic Cell-Penetrating Peptides for Enhanced Penetration of Biological Barriers

Cancer has been a grand challenge in global health. However, the complex tumor microenvironment generally limits the access of therapeutics to deeper tumor cells, leading to tumor recurrence. To conquer the limited penetration of biological barriers, cell-penetrating peptides (CPPs) have been discovered with excellent membrane translocation ability and have emerged as useful molecular transporters for delivering various cargoes into cells. However, conventional linear CPPs generally show compromised proteolytic stability, which limits their permeability across biological barriers. Thus, the development of novel molecular transporters that can penetrate biological barriers and exhibit enhanced proteolytic stability is highly desired to promote drug delivery efficiency in biomedical applications. We have previously synthesized a panel of short cyclic CPPs with aromatic crosslinks, which exhibited superior permeability in cancer cells and tissues compared to their linear counterparts. Here, a concise protocol is described for the synthesis of the fluorescently labeled cyclic polyarginine R8 peptide and its linear counterpart, as well as key steps for investigating their cell permeability.

This protocol describes the synthesis of cyclic cell-penetrating peptides with aromatic cross-links and the evaluation of their permeability across biological barriers.

Cancer has been a grand challenge in global health. However, the complex tumor microenvironment generally limits the access of therapeutics to deeper ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

Characterization of Intra-Cartilage Transport Properties of Cationic Peptide Carriers

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