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>
			<td>Epoxy, Non-corrosive, clear</td>
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		<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>
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		<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 ...

Characterizing the transport properties of therapeutics and their carriers is critical for ensuring effective biological responses. These methods aid in designing optimally charged drug carriers for targeting negatively charged tissues. The main advantage of these techniques is their ability to better predict in vivo therapeutic efficacies through a series of in vitro experiments that characterize solute transport through tissue.Cartilage diseases, such as osteoarthritis, remain untreated due to the inability of drugs to penetrate the dense avascular cartilage matrix, requiring drug carriers to prologue therapeutic efficacy. Begin by using a delicate task wipe to gently remove excess PBS from the surfaces of three millimeter diameter, one millimeter thick cartilage explants. Use a balance to quickly record the wet weight of each explant and immediately place the explants into a PBS bath to prevent dehydration.Next, add 300 microliters of freshly prepared, 30 micromolar fluorescently labeled cationic peptide carrier solution per well to the inner wells of a 96-well plate and use a spatula to add one explant to each well of solution. Fill each of the surrounding wells with 300 microliters of PBS and cover the plate with a lid. Seal the edges of the plate with flexible film to minimize evaporation and place the plate on a shaker in a 37 degree Celsius incubator for 24 hours at 50 revolutions per minute, with a 15 millimeter orbit.At the end of the incubation, transfer the equilibrium bath from each well into individual polypropylene tubes and make serial dilutions from the stock 30 micromolar cationic peptide carrier solution to generate a standard curve. Then, transfer 200 microliters of each solution and standard into individual wells of a black 96-well plate and obtain fluorescence readings of each sample and standard based on the excitation and emission wavelengths of the fluorescent label. To determine the depth of cationic peptide carrier penetration within a cartilage explant, use a scalpel to cut six millimeter diameter, one millimeter thick cartilage explants in half and hydrate the resulting half-disc pieces with proteus inhibitor supplemented PBS.Apply epoxy to the center of a well of a custom-designed one-dimensional transport chamber and secure one half-disk explant within the well with the superficial side of the explant facing the upstream side of the chamber. Remove any excess glue from the well to prevent contact with the diffusion surface area of the cartilage and add 80 microliters of protease inhibitor supplemented PBS to both sides of the explant. Pipette the liquid up and down on one side of the explant to check for leakage to the other side.If no leakage is present, replace the protease inhibitor supplemented PBS from the upstream side with 80 microliters of 30 micromolar fluorescence labeled cationic peptide carrier solution and carefully place the transport chamber into a cell culture dish. Cover the base of the dish with PBS to avoid cationic peptide carrier solution evaporation. Taking care that there is no direct contact between the solutions from the upstream and downstream chambers.Place the covered dish on a shaker to limit particle sedimentation for four or 24 hours at room temperature and 50 revolutions per minute, with a 15 millimeter orbit. At the end of the incubation, remove the explants from the chamber and cut approximately 100 micron thick slices from the center of each explant. Place each slice of explant between a glass slide and a cover slip and hydrate the slices with fresh protease inhibitor supplemented PBS.Fix the slide onto the stage of a confocal microscope and obtain a Z-stack of fluorescent images through the full thickness of the slice at 10X magnification. Open the image file and image J, click Image, and select Stacks and Z Project from the dropdown menu. Then, input the slice numbers from one to the final slice and under Projection Type, select average intensity and click Okay.To assess the non-equilibrium cationic peptide carrier cartilage diffusion rate, assemble each half of the transport chamber, to include one large rubber gasket, one polymethylmethacrylate insert, and one small rubber gasket. Measure the thickness of a cartilage explant, then place the explant in the wells of the plastic insert with the superficial surface facing the upstream chamber and sandwich the two halves together to complete the assembly. Use a wrench to tightly screw the halves together before filling the upstream chamber with two milliliters of protease inhibitor supplemented PBS.Check the downstream chamber for leakage from the upstream chamber. If no leakage is detected, fill the downstream chamber with two milliliters of protease inhibitor supplemented PBS. Add a single mini stir bar to both the up and downstream chambers and place the chamber on a stir plate with the chamber aligned such that the laser from the spectrophotometer is focused towards the center of the downstream chamber.With the signal receiver portion of the spectrophotometer behind the downstream chamber, collect stable, realtime downstream fluorescence emission readings for at least five minutes. After obtaining a stable reading, add a pre-calculated volume of fluorescently labeled cationic peptide carrier stock solution to the upstream chamber to a final bath concentration of three micromolar, and monitor the downstream fluorescent signal while allowing the solute transport to reach a steady increase in slope. Once a steady state has been reached, transfer 20 microliters of solution from the upstream chamber to the downstream chamber for a spike test and collect the real-time downstream fluorescence readings.Too high a positive charge will limit solute penetration to the superficial zone as the carrier binds too strongly to the negatively charged aggrecan groups of the cartilage matrix. Conversely, carriers that are able to take advantage of weak and reversible charge interactions penetrate through the deep zone of tissue. An optimally charged drug carrier, however, would not only penetrate the deep zones of tissue, but would also demonstrate a high intra-tissue uptake, which can be determined using the fluorescence measurements of the equilibrium bath and tissue wet weight, as shown.Non-equilibrium diffusion transport experiments result in a data generated curve with a gradually increasing slope. The initial part of the curve represents solute diffusion through the cartilage as solute matrix binding interactions occur. Once solutes reach the downstream chamber, the slope of the curve increases as the fluorescence readings increase over time.This second part of the curve then reaches a steady slope, representing steady state diffusion. The X-intercept of a tangential line drawn at the steady state portion of the curve indicates the time it takes to reach steady state diffusion or tau lag. Following solution transfer from the upstream to the downstream chamber, a spike in fluorescence is observed, at which point the stabilized fluorescence intensity can be used to correlate the fluorescence to the concentration.The representative effective diffusivity and steady state diffusion values can then be calculated, as indicated. Note that the steady state diffusivity is two orders of magnitude higher than the effective diffusivity as a result of all of the charge-based binding sites being occupied. Thus, at this point, diffusion is based on size and not charge, resulting in similar steady state diffusion values among CPCs of same size.Maintain explant hydration and minimize solution evaporation throughout the experiment to prevent changes in the cartilage morphology and the solution concentration, respectively, and to ensure accurate and reproducible data acquisition. Following the design of an optimally charged drug carrier, various conjugation techniques can be utilized to modify drugs for enhanced tissue targeting and to facilitate evaluation of their biological efficacy.

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