Name: characterization of intra-cartilage transport properties of cationic peptide carriers
Uploaded: 2020-08-10T13:00:00
Description: Watch this Scientific Journal Video about Characterization of Intra-Cartilage Transport Properties of Cationic Peptide Carriers at JoVE.com

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 plu...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avidin+as+a+model+for+charge+driven+transport+into+cartilage+and+drug+delivery+for+treating+early+stage+post-traumatic+osteoarthritis.">Avidin as a model for charge driven transport into cartilage and drug delivery for treating early stage post-traumatic osteoarthritis.</a> Biomaterials. 35 (1), 538-549 (2014).</li><li>Vedadghavami, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cartilage+penetrating+cationic+peptide+carriers+for+applications+in+drug+delivery+to+avascular+negatively+charged+tissues.">Cartilage penetrating cationic peptide carriers for applications in drug delivery to avascular negatively charged tissues.</a> Acta Biomaterialia. 93, 258-269 (2019).</li><li>Mehta, S., Akhtar, S., Porter, R. M., &#214;nnerfjord, P., Bajpayee, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-1+receptor+antagonist+(IL-1Ra)+is+more+effective+in+suppressing+cytokine-induced+catabolism+in+cartilage-synovium+co-culture+than+in+cartilage+monoculture.">Interleukin-1 receptor antagonist (IL-1Ra) is more effective in suppressing cytokine-induced catabolism in cartilage-synovium co-culture than in cartilage monoculture.</a> Arthritis Research &amp; Therapy. 21 (1), 238 (2019).</li><li>Vedadghavami, A., Zhang, C., Bajpayee, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overcoming+negatively+charged+tissue+barriers:+Drug+delivery+using+cationic+peptides+and+proteins.">Overcoming negatively charged tissue barriers: Drug delivery using cationic peptides and proteins.</a> Nano Today. 34, 100898 (2020).</li><li>Young, C. C., Vedadghavami, A., Bajpayee, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioelectricity+for+Drug+Delivery:+The+Promise+of+Cationic+Therapeutics.">Bioelectricity for Drug Delivery: The Promise of Cationic Therapeutics.</a> Bioelectricity. , (2020).</li><li>Felson, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritis+of+the+knee.">Osteoarthritis of the knee.</a> New England Journal of Medicine. 354 (8), 841-848 (2006).</li><li>Wieland, H. A., Michaelis, M., Kirschbaum, B. J., Rudolphi, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritis+-+An+untreatable+disease.">Osteoarthritis - An untreatable disease.</a> Nature Reviews Drug Discovery. 4 (4), 331-344 (2005).</li><li>Martel-Pelletier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+osteoarthritis.">Pathophysiology of osteoarthritis.</a> Osteoarthritis and Cartilage. 7 (4), 371-373 (1999).</li><li>Sophia Fox, A. J., Bedi, A., Rodeo, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basic+science+of+articular+cartilage:+Structure,+composition,+and+function.">The basic science of articular cartilage: Structure, composition, and function.</a> Sports Health. 1 (6), 461-468 (2009).</li><li>Chevalier, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraarticular+injection+of+anakinra+in+osteoarthritis+of+the+knee:+A+multicenter,+randomized,+double-blind,+placebo-controlled+study.">Intraarticular injection of anakinra in osteoarthritis of the knee: A multicenter, randomized, double-blind, placebo-controlled study.</a> Arthritis Care and Research. 61 (3), 344-352 (2009).</li><li>Cohen, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+randomized,+double-blind+study+of+AMG+108+(a+fully+human+monoclonal+antibody+to+IL-1R1)+in+patients+with+osteoarthritis+of+the+knee.">A randomized, double-blind study of AMG 108 (a fully human monoclonal antibody to IL-1R1) in patients with osteoarthritis of the knee.</a> Arthritis Research and Therapy. 13 (4), 125 (2011).</li><li>Evans, C. H., Kraus, V. B., Setton, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+intra-articular+therapy.">Progress in intra-articular therapy.</a> Nature Reviews Rheumatology. 10 (1), 11-22 (2014).</li><li>He, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-arm+Avidin+nano-construct+for+intra-cartilage+delivery+of+small+molecule+drugs.">Multi-arm Avidin nano-construct for intra-cartilage delivery of small molecule drugs.</a> Journal of Controlled Release. 318, 109-123 (2020).</li><li>Bajpayee, A. G., Scheu, M., Grodzinsky, A. J., Porter, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rabbit+model+demonstrates+the+influence+of+cartilage+thickness+on+intra-articular+drug+delivery+and+retention+within+cartilage.">A rabbit model demonstrates the influence of cartilage thickness on intra-articular drug delivery and retention within cartilage.</a> Journal of Orthopaedic Research. 33 (5), 660-667 (2015).</li><li>Bajpayee, A. G., Quadir, M. A., Hammond, P. T., Grodzinsky, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charge+based+intra-cartilage+delivery+of+single+dose+dexamethasone+using+Avidin+nano-carriers+suppresses+cytokine-induced+catabolism+long+term.">Charge based intra-cartilage delivery of single dose dexamethasone using Avidin nano-carriers suppresses cytokine-induced catabolism long term.</a> Osteoarthritis and Cartilage. 24 (1), 71-81 (2016).</li><li>Zhang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avidin-biotin+technology+to+synthesize+multi-arm+nano-construct+for+drug+delivery.">Avidin-biotin technology to synthesize multi-arm nano-construct for drug delivery.</a> MethodsX. , 100882 (2020).</li><li>Wagner, E. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avidin+grafted+dextran+nanostructure+enables+a+month-long+intra-discal+retention.">Avidin grafted dextran nanostructure enables a month-long intra-discal retention.</a> Scientific Reports. 10.1, 1-14 (2020).</li><li>Troeberg, L., Nagase, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteases+involved+in+cartilage+matrix+degradation+in+osteoarthritis.">Proteases involved in cartilage matrix degradation in osteoarthritis.</a> Biochimica et Biophysica Acta - Proteins and Proteomics. 1824 (1), 133-145 (2012).</li><li>Kirk, T. B., Wilson, A. S., Stachowiak, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+dehydration+on+the+surface+morphology+of+articular+cartilage.">The effects of dehydration on the surface morphology of articular cartilage.</a> Journal of Orthopaedic Rheumatology. 6 (2-3), 75-80 (1993).</li><li>Ateshian, G. A., Maas, S., Weiss, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solute+transport+across+a+contact+interface+in+deformable+porous+media.">Solute transport across a contact interface in deformable porous media.</a> Journal of Biomechanics. 45 (6), 1023-1027 (2012).</li><li>Arbabi, V., Pouran, B., Weinans, H., Zadpoor, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphasic+modeling+of+charged+solute+transport+across+articular+cartilage:+Application+of+multi-zone+finite-bath+model.">Multiphasic modeling of charged solute transport across articular cartilage: Application of multi-zone finite-bath model.</a> Journal of Biomechanics. 49 (9), 1510-1517 (2016).</li><li>Arbabi, V., Pouran, B., Zadpoor, A. A., Weinans, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+and+finite+element+protocol+to+investigate+the+transport+of+neutral+and+charged+solutes+across+articular+cartilage.">An experimental and finite element protocol to investigate the transport of neutral and charged solutes across articular cartilage.</a> Journal of Visualized Experiments. 2017 (122), (2017).</li><li>Sampson, S. L., Sylvia, M., Fields, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+dynamic+loading+on+solute+transport+through+the+human+cartilage+endplate.">Effects of dynamic loading on solute transport through the human cartilage endplate.</a> Journal of Biomechanics. 83, 273-279 (2019).</li><li>Bajpayee, A. G., Scheu, M., Grodzinsky, A. J., Porter, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrostatic+interactions+enable+rapid+penetration,+enhanced+uptake+and+retention+of+intra-articular+injected+avidin+in+rat+knee+joints.">Electrostatic interactions enable rapid penetration, enhanced uptake and retention of intra-articular injected avidin in rat knee joints.</a> Journal of Orthopaedic Research : Official Publication of the Orthopaedic Research Society. 32 (8), 1044-1051 (2014).</li><li>Bajpayee, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+intra-cartilage+delivery+of+low+dose+dexamethasone+using+a+cationic+carrier+for+treatment+of+post+traumatic+osteoarthritis.">Sustained intra-cartilage delivery of low dose dexamethasone using a cationic carrier for treatment of post traumatic osteoarthritis.</a> European Cells &amp; Materials. 34, 341-364 (2017).</li><li>Malda, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+Mice,+Men+and+Elephants:+The+Relation+between+Articular+Cartilage+Thickness+and+Body+Mass.">Of Mice, Men and Elephants: The Relation between Articular Cartilage Thickness and Body Mass.</a> PLoS One. 8 (2), 57683 (2013).</li><li>Frisbie, D. D., Cross, M. W., McIlwraith, C. 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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&#34; ID, 0.312&#34; 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&#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>
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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 ...

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