Liposomes Enhance Dye Localization within the Mammary Ducts of

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Liposomes Enhance Dye Localization within the Mammary Ducts of Porcine Nipples Samantha L Kurtz, and Louise B Lawson Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00037 • Publication Date (Web): 05 Mar 2019 Downloaded from http://pubs.acs.org on March 6, 2019

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Molecular Pharmaceutics

Liposomes Enhance Dye Localization within the Mammary Ducts of Porcine Nipples

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Samantha L. Kurtz a,b, Louise B. Lawson a*

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aDepartment

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Orleans, LA 70112 USA

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bBioinnovation

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Orleans, LA 70118 USA

of Microbiology and Immunology, Tulane University School of Medicine, New

Ph.D. Program, Tulane University School of Science and Engineering, New

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*

Corresponding author: Mailing address: Department of Microbiology and Immunology, Tulane

University School of Medicine, 1430 Tulane Ave (8638), New Orleans, LA 70112, USA. Tel.: (504)988-2204; Fax: (504)988-5144 E-mail Address: [email protected] (L.B. Lawson)

ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

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Transductal and transepidermal diffusion are two distinct penetration routes of molecules

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administered via the nipple. To improve the therapeutic potential of this drug administration

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technique, drug penetration into the mammary ducts should be maximized, which may be

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accomplished through design optimization of drug delivery vehicles. In this study, we evaluated

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liposomes, ranging in size from 100 to 3,000 nm, to improve ductal penetration of model

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fluorescent dyes using fluorescence microscopy and image analysis. Liposomes encapsulating

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either a model fluorescent lipophilic dye, nile red, or hydrophilic dye, sulforhodamine B, were

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applied topically on porcine nipples for 6 hours in vitro. Liposome encapsulation of

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sulforhodamine B significantly reduced the total amount of dye penetrating the nipple, while

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penetration of liposome-encapsulated nile red varied depending on vesicle size, as compared to

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their solution controls. However, the fluorescence intensity localized at the ductal epithelium

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was higher at extended nipple depths in tissues treated with liposomes versus dye solutions,

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suggesting a higher concentration of dye penetrating the nipple via the ducts. In contrast, the

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fluorescence intensity measured at the stratum corneum was reduced (sulforhodamine B) or

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unchanged (nile red) in nipples treated with liposomes versus dye solutions, suggesting a

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decrease or no change in dye penetration of the nipple via the stratum corneum. Furthermore,

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the limited penetration distance into the connective tissue beyond the ductal epithelium for both

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liposome-encapsulated nile red and sulforhodamine B suggests that liposomes remain intact

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over the 6-hour duration of this study when penetrating through the ducts and enhance retention

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within the ductal lumen. However, the varied penetration profiles into the connective tissue

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beyond the stratum corneum between liposome-encapsulated nile red and sulforhodamine B

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suggests that the liposomes destabilize when penetrating the outer tissues layers of the nipple.

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Overall, liposomes, regardless of size, improved penetration into and retention within the

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mammary ducts, while limiting penetration into the stratum corneum, indicating their capacity to

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target the mammary ductal network.


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KEYWORDS

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Transpapillary drug delivery

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Liposomes

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Nanoparticle

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Breast cancer

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Mammary ducts

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Breast ducts

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INTRODUCTION In the United States, over 160,000 women annually are diagnosed with either pre-

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cancerous or non-invasive breast cancer lesions, including atypical hyperplasia and carcinoma

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in situ 1, 2. Despite the local nature of these conditions, systemic therapy, radiation or life-altering

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surgery, each of which results in a plethora of side effects and psychological detriments, are the

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only therapeutic options. However, targeted, local pharmaceutical administration within the

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epithelial ductal and lobular lining of the mammary ductal network could maximize drug

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exposure where 95% of breast cancers originate, in turn limiting exposure of healthy tissues

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and, as a result, reducing adverse side effects 3.

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The mammary ductal network is directly accessible via 5 to 8 ductal channels that

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originate in the human nipple 4. Two drug administration strategies, intraductal injection and

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transpapillary diffusion, capitalize on these orifices to provide an entry point for molecules, yet

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do so with markedly different approaches. Intraductal injection involves cannulation of one or

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more orifice using a catheter to instill a pharmaceutical solution within the mammary channels 5.

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However, the requirement of a surgical professional and the invasiveness of this technique

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precludes the option of self-administration in the at-home setting. On the contrary, transpapillary

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diffusion is a non-invasive technique that could allow self-administration of a cream or patch

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topically on the nipple. The feasibility and mechanistic aspects of this delivery method have

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been evaluated in vitro and in vivo using a variety of molecules 6-10. However, while the ductal

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orifices are a primary penetration route, molecules also permeate across the exposed stratum

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corneum and diffuse into the connective tissue of the nipple to a varying degree, depending on

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the physicochemical attributes of the administered drug. While this may be advantageous for

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some therapeutic purposes, maximizing penetration into the ductal channels is the optimal

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target for treatment of non-invasive breast lesions.

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To improve the therapeutic potential of transpapillary diffusion, tailored design of a drug delivery vehicle could maximize transport into and through the mammary ducts. Although a


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number of physicochemical characteristics are likely to influence transport, molecular weight

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and hydrophilicity are two known properties that directly impact diffusion 7, 8, 10. For instance,

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molecular weight is inversely related to penetration into the nipple. Molecules smaller than 0.5

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kDa penetrate up to 35% of the total applied dose after 48 hours, while a 10 kDa

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macromolecule penetrates only 3%, and a 50 kDa macromolecule less than 1% 7, 10. While the

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extent of ductal penetration and retention was not assessed for all molecules, this critical factor

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is also impacted by drug encapsulation, as demonstrated by pharmacokinetic analysis of

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intraductally administered agents 3, 5. For small drug molecule carboplatin, the plasma drug

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exposure following intraductal or intravenous injection was the same, indicating minimal

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retention within the mammary ducts and no difference in systemic exposure 5. However,

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intraductal injection of a 100 nm liposome encapsulating doxorubicin reduced the plasma drug

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concentration five to ten-fold as compared to intravenous injection 3, 5, suggesting that drug

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carriers, liposomes specifically, limit diffusion out of the mammary ducts and into blood

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circulation, thereby diminishing the systemic exposure. Enhanced retention within the ducts has

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been demonstrated with intraductally injected vesicles as small as 5 -10 nm and as large as 100

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nm 3, 5, 11. However, passive diffusion of drug carriers and their retention within the mammary

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channels has yet to be characterized.

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In addition to size, the hydrophilicity of a drug molecule or its carrier also impacts

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transpapillary diffusion. Lipophilic small molecules localize within the mammary ducts, while the

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dominant penetration pathway for hydrophilic molecules is the stratum corneum 8, 10. Similarly,

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lipid-based drug transport vesicles, such as liposomes, provide enhanced delivery of both

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hydrophilic and hydrophobic molecules into skin orifices 12 and therefore are likely to enhance

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diffusion via the innate ductal channels as well. A liposome is a multilayered vesicle composed

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of amphiphilic phospholipids capable of incorporating either hydrophilic molecules within the

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aqueous core or hydrophobic molecules within the lipid bilayer 13. The physicochemical



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properties of these biocompatible vesicles, such as size and charge, are versatile and have, in

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part, attributed to their clinical success 13, 14.

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In the present study, we investigated diffusion of liposomes through porcine nipples in

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vitro. Liposomes encapsulated either a model fluorescent lipophilic dye, nile red (NR), or

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fluorescent hydrophilic dye, sulforhodamine B (SRB). Total dye penetration was quantified using

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fluorimetry. For evaluation of the specific transport pathways, dye penetration into the nipple via

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the ducts or stratum corneum was quantified using fluorescence image analysis. Tissue

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sections from sequential nipple depths were analyzed to elucidate the impact of liposomes on

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both ductal and stratum corneum penetration and retention.

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EXPERIMENTAL SECTION

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Materials

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Acetone, Cytoseal60, ethanol, isoamyl alcohol, nile red (NR), phosphate buffered saline

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(PBS), sulforhodamine B (SRB) were purchased from ThermoFisher. Biosol was purchased

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from National Diagnostics. Egg phosphatidylcholine (EPC) was purchased from Avanti Polar

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Lipids, Inc. Porcine abdominal skin tissue was supplied by a local market (Verdun Farms Meat

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Market; Raceland, LA).

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Liposome preparation Liposomes were prepared using the thin film hydration method. A 6.25 mg/mL solution of

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EPC dissolved in chloroform was added to a round-bottom flask. For liposomes encapsulating

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model dye NR, a 1 mM solution of NR solubilized in ethanol was also added to the flask. Then,

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the organic solvent was removed by rotary evaporation for 2 hours. The subsequent dry lipid

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film deposited on the flask was hydrated with PBS by vortexing for 2 minutes. For liposomes

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encapsulating model dye SRB, the dye was dissolved at 10 mM in PBS prior to lipid film

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hydration. After lipid hydration, some preparations were extruded through polycarbonate


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121

membranes to reduce liposome size. Extruded liposomes were passed through two membranes

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of either 400 nm or 100 nm pore sizes 21 times at 45°C and then were then left overnight at 4°C

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to stabilize. Meanwhile, liposome suspensions that were not extruded (unextruded) were

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immediately stored at 4°C to stabilize overnight. Finally, the liposome dispersions were diluted

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to 2 mL in PBS and ultracentrifuged (Beckman TL-100 Ultracentrifuge with a Beckman TLS 55

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rotor) to remove unencapsulated dye. The unextruded liposome preparations were centrifuged

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at 53,000xg at 4°C for two hours, while the extruded liposomes were centrifuged at 258,000xg

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at 4°C for 24 hours. The supernatant was removed and liposome pellets were resuspended in 2

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mL PBS.

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Nanoparticle size and distribution The size, polydispersity index (PDI), and zeta potential of liposomes were measured by

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dynamic light scattering using a NanoBrook 90Plus PALS Instrument. Liposome suspensions

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were diluted 5 to 12-fold in water prior to measurement. The reported data is an average of the

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particle intensity values from quintuplicate measurements of three individual liposome

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preparations.

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Encapsulation efficiency Samples (100 µL) of the liposomal solution were taken prior to (Ctotal) and post

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(Cencapsulated) ultracentrifugation. Liposomes were lysed by mixing the solutions with ethanol at a

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1:19 (v:v) ratio. Fluorescence was measured on a Synergy H1 fluorimeter at 550/630

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excitation/emission for NR and 560/590 excitation/emission for SRB. The dye concentration was

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then calculated by interpolating from an established calibration curve. The encapsulation

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efficiency (EE) was calculated as: EE%= (Cencapsulated/Ctotal) x 100.

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In vitro nipple permeation



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All experiments were performed using porcine nipple tissue, which is a well-suited model

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of the human nipple 8. Upon receipt, nipples were separated from surrounding abdominal tissue

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and stored at -20°C. Prior to testing, nipples were thawed, rinsed, and any remaining underlying

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dermal fat tissue was removed. The nipple was then mounted on a jacketed Franz diffusion cell

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(surface area 0.64cm2; Permegear) with the tip of the nipple in direct contact with the donor

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compartment similar to previous reports 10. The receptor compartment was filled with 5 mL of

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PBS, maintained at 37°C using a circulating water bath and consistently stirred to maintain a

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homogeneous mixture. A 400 µL liposome dispersion or dye solution was applied topically for 6

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hours. For the dye solutions, a 0.1 mM of NR solubilized in ethanol or a 0.2 mM solution of SRB

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solubilized in PBS was applied. Ethanol was used as a solvent for NR due to its low solubility

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and fluorescence quenching in aqueous solvents 15. After treatment, the excess donor

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compartment liposome formulations were collected and diluted 12-fold in distilled water prior to

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size measurement using DLS, as reported above. For all treatments, the tissue surface was

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rinsed with 3mL of ethanol and the tissue was then stored at -80°C until sectioning. To avoid

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redistribution of the fluorescent labels, tissue samples were not fixed or cryo-embedded prior to

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freezing.

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Dye retention in nipple Nipples were coronally sectioned from tip to base in 100 µm increments on a cryostat

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(Leica CM1860). Groups of ten sequential sections (1 mm of tissue) were collected into a pre-

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weighed tube. The tube was weighed post-addition of tissue and then a solvent was added for

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dye extraction. For tissue containing NR, 350 µL of 6:1 isoamyl alcohol:acetone (v:v) was added

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to each tube and incubated for a minimum of 30 minutes. For tissue containing SRB, 400 µL of

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Biosol was added to each tube and incubated at 37C for at least one hour. To generate a

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standard curve, known concentrations of NR or SRB were added to the appropriate dilution

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medium and serially diluted across a black-walled, flat-bottomed 96-well plate. Samples from


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each respective tube were added to the plate and the fluorescence was determined on a

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Synergy H1 fluorimeter at 550/630 excitation/emission for NR and 560/590 excitation/emission

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for SRB.

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Fluorescence imaging For qualitative comparison of liposome and free dye penetration, the spatial distribution

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of dye in the nipple was captured using fluorescence imaging. During cryo-sectioning, 20-µm

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sections were taken every 500 µm, mounted on glass slides, and incubated at 37C overnight

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prior to sealing with Cytoseal60. Fluorescence images were taken on a Zeiss Axioplan 2 with a

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Cy3 filter set (Chroma HQ 41007). The exposure time and image normalization were

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standardized to minimize pixel intensity variation between tissue sections due to parameter

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settings. A single micrograph of an entire nipple cross section was generated by imaging

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consecutive tissue regions and stitching images together using SlideBook 6 Software (Intelligent

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Imaging Software).

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Image analysis

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For quantitative comparison of liposome and free dye penetration, fluorescence

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micrographs were analyzed using ImageJ software 16. Montage images of the entire nipple

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cross section were used for stratum corneum image analysis, while 10x images of the

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mammary ducts were used for ductal image analysis. A fluorescence intensity profile was

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generated as a function of distance normal to the tissue edge for quantification of penetration

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via the stratum corneum or normal to the ductal edge for quantification of penetration via the

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ducts. This facilitated analysis of the fluorescence intensity as a function of penetration distance

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(xy plane) at nipple depths ranging from 1.0 to 5.0 mm (z coordinate) (Figure 1). Between five

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and eight plot profile lines were averaged per localized region (stratum corneum or duct) per

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nipple (n=6).



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Figure 1. Determination of fluorescence intensity profiles using ImageJ Plot Profile tool.

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Sequential images of an entire porcine nipple cross section were compiled to create a single

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fluorescence micrograph (A). The line tool in ImageJ was then drawn across the stratum

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corneum or ductal lumen (B, blue line) to generate the fluorescence pixel intensity as a function

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of distance (C). For stratum corneum penetration, eight intensity profiles were averaged per

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tissue section. For ductal penetration, five intensity profiles were averaged per duct.

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Statistical Analysis

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All statistical analysis was performed using GraphPad Prism software version 7 (San Diego,

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CA). A one-way or two-way ANOVA with Tukey’s multiple comparisons post-test was used to

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analyze the total amount of dye deposited or to analyze the effect of treatment by depth,

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respectively. Results were deemed significant when p ≤ 0.05.

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RESULTS AND DISCUSSION

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Physicochemical characterization of liposomes

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We employed two fluorescent dyes, NR and SRB, to assess the impact of liposome

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encapsulation on transpapillary permeation. The fluorescent dyes are similar in molecular

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weight (NR MW = 318.37 Da vs SRB MW = 558.6 Da) but differ in their lipophilicity as indicated


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by their logP values (NR logP = 5 vs SRB logP = -2) 10. Liposomes encapsulating either NR or

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SRB had similar average diameters of approximately 3,000 nm, 265 nm and 120 nm for

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preparations that were unextruded, extruded through a 400 nm membrane, or extruded through

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a 100 nm membrane, respectively (Table 1). The PDI was highest for unextruded liposomes,

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while extruded liposomes were nearly homogeneous with PDI values of 0.232 or less. For lipid-

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based drug delivery formulations, PDI values less than 0.3 are considered monodisperse 17. The

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zeta potential of all formulations ranged from -0.664 – 2.531 mV, which is consistent with

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previous results of EPC-composed liposomes 18, 19. Because the zeta potentials fall within the -

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10 to 10 mV range, these vesicles are considered neutral 20, 21. NR had a higher encapsulation

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efficiency compared to SRB (91.11-96.62% vs 57.13-69.61%), likely due to their solubility

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differences and partitioning characteristics. Based on their logP values, these model dyes are

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almost exclusively located within the transmembrane region (NR) or aqueous interior (SRB).

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Passive encapsulation of hydrophilic agents is typically less efficient than lipophilic agents due

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to their solubility in both the continuous phase and aqueous liposomal interior 14, 22, 23. The final

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dye concentration of each liposome dispersion following removal of unencapsulated dye is

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presented in Table 1. While the particle concentration was not determined, the total amount of

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lipids used and subsequent dilutions during liposome ultracentrifugation were standardized to

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minimize the potential impact of lipid content on transpapillary penetration.

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Table 1. Physical characterization of NR or SRB loaded liposomes (Mean  SEM). Diameter (nm) PDI Zeta Potential (mV) EE% [dye] (mM) NR Liposomes 3340  951.4 0.536  0.097 0.207  0.375 96.62  1.41 0.132  0.023 276.4  25.87 0.232  0.022 -0.664  0.736 93.43  4.09 0.076  0.008 123.6  18.1 0.127  0.031 -0.093  0.067 91.11  4.36 0.066  0.004 SRB Liposomes 2875  584.3 1.214  0.490 2.531  0.752 69.61  1.89 0.187  0.013 255.2  7.79 0.166  0.019 0.801  2.110 57.13  3.98 0.141  0.023 117.6  16.25 0.146  0.035 -0.115  0.023 57.73  1.71 0.177  0.026 Each measurement is an average of at least 3 individual liposome preparations. PDI, polydispersity index; EE, encapsulation efficiency; NR, nile red; SRB, sulforhodamine B



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Effect of dye hydrophilicity and liposome size on penetration To understand the influence of liposome encapsulation on transpapillary diffusion, either

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liposomes encapsulating the model fluorescent dyes or the dyes in solution were applied

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topically on porcine nipples in vitro for 6 hours. In an effort to keep the overall lipid concentration

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consistent, dilutions during liposome preparation were standardized and the total volume

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applied topically (400 µL) was constant. Because of the differences in dye encapsulation

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efficiency, the dye concentration did vary among treatments for each dye (Table 1). Therefore,

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the quantity of dye retained within the tissue (Figure 2) is represented as both as a percent of

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dye applied, accounting for dye discrepancies, as well as a function of tissue weight.

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The penetration of liposome-encapsulated dye or dye solutions into the nipple revealed

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statistically significant differences depending on the fluorescent dye and vesicle size. The total

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amount of NR penetrating the nipple ranged from 1.28 - 5.56% of the total applied dye after 6

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hours (Figure 2A), while the total amount of SRB penetrating the nipple ranged from 6.19 -

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29.43% (Figure 2B). As previously demonstrated 10, the solution of SRB more efficiently

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penetrated the nipple than the solution of NR (29.34% vs 2.65%).

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Figure 2. Dye retention in porcine nipple after in vitro diffusion for 6 hours. NR (A-B) or SRB (C-

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D) is reported as total percent applied (A,C) or delineated by depth (B,D) when applied in

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solution or encapsulated within liposomes. Data is represented as mean  SEM (n=6).

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Statistical analysis was performed with one-way ANOVA (Retained Dye %) or two-way ANOVA

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(Retained Dye nmol/g) with Tukey’s multiple comparisons post-test. p ≤ 0.05 was considered

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significant and is denoted using the following indicators: a- solution vs unextruded, b- solution vs

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400 nm extruded, c- solution vs 100 nm extruded, d- unextruded vs 400 nm extruded, e-

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unextruded vs 100 nm extruded, f- 400 nm extruded vs 100 nm extruded.

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The influence of liposome encapsulation on NR penetration depended on vesicle size. The smallest liposome size (123.6  18.1 nm) deposited more dye than the NR solution, while the



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largest liposome size (3340  951.4 nm) deposited less dye than the solution control. The

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intermediate liposome size (276.4  25.87 nm) and the NR solution penetrated nearly equivalent

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amounts. However, a more distinct trend was found for SRB-treated tissue. Liposome

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encapsulation significantly reduced SRB penetration by as much as 4.7-fold as compared to the

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SRB solution. These results are consistent with previous reports of transdermal penetration with

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liposomes of similar size and formulation. For 100 nm soy phosphatidlcholine liposomes, the

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greatest reduction in penetration was found for the most hydrophilic molecule tested,

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amphotericin B (logP=0.8). Meanwhile, more lipophilic small molecules, imiquimod (logP=2.7)

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and indole (logP=2.14), had either slightly reduced or no penetration change upon liposome

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encapsulation 24. Transdermal penetration, and likely transpapillary diffusion, however, is highly

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influenced by liposome composition. For example, incorporation of membrane stabilizers, such

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as cholesterol, or surface functionalizers, such as polyethylene glycol, can enhance transdermal

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penetration of encapsulated hydrophilic or lipophilic molecules 25, 26.

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Among the liposome formulations, vesicle size was inversely related to penetration amount.

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The largest vesicles (2875 – 3340 nm) deposited the least amount of dye, while the smallest

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vesicles (117.6 – 123.6 nm) deposited the most NR and SRB. Similar results were found for

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penetration into the skin. In previous reports, within both the stratum corneum and dermis, the

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smaller the liposome, the greater the penetration 27. However, in the current study, the variation

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between each liposomal treatment was less than 5%, suggesting only a slight size effect for

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EPC liposomes administered via the transpapillary route.

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When delineating dye penetration by depth, the largest differences among treatments were

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found in the top of the nipple (Figure 2B & 2D). At the uppermost tissue section (0-1 mm),

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ethanolic NR deposited at least 3.6-fold less dye than liposomal NR. As nipple depth increased,

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the amount of NR deposited by the ethanolic solution slowly increased, reaching a maximum

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between 2-5 mm in nipple depth before decreasing for the bottom-most 3 mm. Retention of NR


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administered via liposome formulations, however, rapidly and consistently decreased over the

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entire depth of the nipple. Dye retention as a function of depth for the NR solution is inconsistent

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with our previous study of an ethanolic NR solution 10, potentially due to the solvents in the

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receptor compartment of the Franz diffusion cell. In the current study, the receptor compartment

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was filled with PBS, whereas previously, receptor compartment contained 100% ethanol.

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Therefore, the differing retention profile in the current study is potentially due to the absorption

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of ethanol into the nipple from the donor compartment and its interface with PBS from the

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receptor compartment, resulting in an increased flux and dye content at the ethanol:PBS

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cosolvent boundary within the tissue 28. Furthermore, in treatments where the solvent in the

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donor and receptor compartments are the same, such as in the earlier study and with the

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liposome formulations and SRB solution in the current study, the dye retention profiles are

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consistent. For the hydrophilic dye, penetration of liposome-encapsulated SRB is over 3.8-fold

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lower than the SRB solution at a depth of 0-1 mm (Figure 2D). As depth increases, both the

308

amount of dye retained and the impact of treatment formulation quickly tapered; however, a

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significant difference was found up to 5.0 mm in depth.

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Among the liposome treatments, a consistent vesicle size effect was found such that the

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smallest liposomes deposited the most dye at each depth range, while the largest vesicles

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deposited the least NR or SRB. The inverse relationship between vesicle size and dye retention

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across tissue depths (Figure 2B and 2D) is in agreement with total dye penetration (Figure 2A

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and 2C).

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After topical application in vitro for 6 hours, no dye was detected in the receptor

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compartment, indicating that none of the treatments facilitated dye diffusion across the entire

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nipple to a detectable level. This is consistent with previous findings that the lag time across the

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nipple ranges from 13-20 hours 7, 8, 10.

319 320

Impact of Topically Applied Liposomes on Transport Pathways through the Nipple



321

Our previous study illustrates two dominant penetration pathways of transpapillary

322

diffusion, transepidermal and transductal 10. To understand the impact of liposomes on each

323

penetration pathway, image analysis was performed on fluorescence micrographs of liposome-

324

and solution-treated nipple sections. A fluorescence intensity profile was generated as a

325

function of penetration distance radially from the ductal lumen into the connective tissue to

326

assess ductal penetration, or from the stratum corneum into the connective tissue to assess

327

transepidermal penetration. Analysis performed at multiple nipple depths elucidated the impact

328

of liposome encapsulation on penetration depth (z coordinate) in addition to penetration

329

distance (xy plane). Representative fluorescence micrographs of nipple cross-sections following

330

diffusion of liposome-encapsulated dye or dye solutions are in supplementary Figure S1 (NR)

331

and Figure S2 (SRB).

332 333 334

Liposome Dye Penetration via the Mammary Ducts The contours of the ductal fluorescence intensity curves show similar trends across

335

tissue depths when comparing NR (Figure 3A) and SRB (Figure 3B) penetration. All liposome

336

formulation fluorescence profiles had a sharp peak at a distance of approximately 15 µm from

337

the edge of the duct followed by a sharp decline, reaching near zero fluorescence intensity at

338

100 µm. In contrast, the fluorescence intensity profiles of dye solutions (both NR and SRB) had

339

a sharp peak followed by marginal, if any, decline, exhibiting detectable fluorescence at 100 µm

340

penetration distance. For example, at 1.0 mm tissue depth, both the NR and SRB solution

341

treated nipple sections had over 3.7-fold higher fluorescence intensity at a distance of 100 µm

342

from the ductal lumen compared to the liposomal treatments. The stark contrast in penetration

343

distance between liposome- or solution-treated tissue suggests that the liposomes limited

344

diffusion from the ductal epithelium into the connective tissue.

345 346


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347 348

Figure 3. Representative fluorescence micrographs of mammary ducts and fluorescence

349

intensity profiles for liposome- or solution-deposited dye. Porcine nipples were treated with NR

350

(A) or SRB (B) either in solution or encapsulated within liposomes in vitro for 6 hours. Each

351

curve is an average of 5 ROIs per duct (n=6-12). White scale bars in lower right corner of

352

images represent 100 µm.



353 354

As nipple depth increased, the penetration distance of the dye solutions gradually

355

decreased. By 2.0 mm for SRB and 3.0 mm for NR, there was no detectable difference at 100

356

µm penetration distance between liposome- or solution-treated tissue. However, the difference

357

near 15 µm penetration distance, where the peak fluorescence intensity occurs, increased with

358

nipple depth. NR treated tissue exhibited the highest peak fluorescence intensities at 1.0 mm in

359

depth, which then gradually declined as nipple depth increased (Figure 4A). In general,

360

liposome encapsulated NR had higher peak fluorescence intensities at the ductal edge

361

compared to the NR solution, however the only significant difference found was between the

362

ethanolic solution and 123.6 nm liposome within the 2.5-3.5 mm depth range (0.0132 < p
255.2 nm > 117.6 nm. For NR



392

liposomes, the ductal peak fluorescence intensities followed the order 123.6 nm > 3340 nm >

393

276.4 nm. The variability with regard to vesicle size and ductal penetration could be due to the

394

chosen size range tested and/or biologically innate variability within ductal channels. For

395

transfollicular drug delivery, polymeric nanoparticles ranging in diameter from 643 nm to 1,500

396

nm penetrated deeper than smaller or larger vesicle sizes 12, 31, 32; this range in vesicle size

397

overlaps our medium (255.2 – 276.4 nm) and large liposome sizes (2875 – 3340 nm).

398

Furthermore, consistent with our results, minor penetration depth differences were found

399

between the follicular penetration of liposomes or nanoparticles ranging from 231 to 449 nm or

400

nanoparticles larger than 900 nm 12, 31. Another possible explanation for the indistinct optimal

401

liposome size for maximal ductal penetration depth is the variable anatomy of the channels

402

themselves. The peak fluorescence intensity of each duct was highly variable, with the

403

exception of the SRB solution control (Figure S4). The basis of this variability is attributable to a

404

variation in the size of ductal orifices and/or natural substances blocking the ductal orifice or

405

channel.

406 407 408

Liposome Dye Penetration via the Stratum Corneum The contours of the stratum corneum fluorescence intensity profiles are slightly different

409

across tissue depths when comparing NR (Figure 5A) and SRB (Figure 5B) diffusion. For NR-

410

treated tissue, at 1.0 mm in depth there was marginal difference between the stratum corneum

411

fluorescence intensity profile of liposome-mediated versus solution penetration. Each profile had

412

a sharp peak approximately 50 µm from the edge of the stratum corneum followed by a sharp

413

decline, reaching near zero fluorescence intensity at a penetration distance of 250 µm.

414

However, as nipple depth increased, distinctions between the penetration distance of liposome-

415

encapsulated NR versus the NR solution became apparent. Liposome-encapsulated NR

416

maintained their fluorescence intensity contours up to 5.0 mm in nipple depth, only reaching a

417

penetration distance of 250 µm into the connective tissue. On the other hand, the penetration


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418

distance of the NR solution increased as nipple depth increased. By 2.0 mm in nipple depth,

419

there was detectable fluorescence at a penetration distance of 500 µm. By 5.0 mm in nipple

420

depth, the fluorescence intensity at a penetration distance of 500 µm was almost 3-fold higher

421

than at 1 mm, with over a 20-fold difference between liposome-encapsulated NR and the NR

422

solution.

423



424

Figure 5. Representative fluorescence micrographs of the stratum corneum and fluorescence

425

intensity profiles following in vitro diffusion of fluorescent dyes in solution or within liposomes.

426

Porcine nipples were treated with NR (A) or SRB (B) either in solution or encapsulated within

427

liposomes in vitro for 6 hours. Each curve is an average of 8 ROIs per tissue section (n=3-6).

428

White scale bars in lower right corner of images represents 500 m.

429 430

For SRB-treated tissue, there was also a reduction in penetration distance between

431

liposome-encapsulated SRB and the SRB solution (Figure 5B), although to a lesser extent than

432

NR-treated nipples. At 1.0 mm in depth, the fluorescence intensity of the SRB solution at a 500

433

µm penetration distance was at least 2.9-fold greater than any of the liposome sizes. At further

434

nipple depths, the difference between the fluorescence intensity at a 500 µm penetration

435

distance between SRB solution and SRB liposomes was maintained.

436

The variable impact of NR and SRB liposome encapsulation on the penetration distance

437

via the stratum corneum suggests the vesicles destabilize at or near the surface of the tissue.

438

For NR, liposome encapsulation minimally reduced the fluorescence intensity at 500 µm

439

penetration distance between 1.4- to 4.0-fold at 1.0 mm depth, but at 5.0 mm in nipple depth,

440

the reduction was 32.7- to 147.0-fold. However, SRB liposome encapsulation maintained a

441

reduction in the fluorescence intensity at 500 µm penetration distance at all nipple depths (2.9-

442

to 17.4-fold at 1.0 mm versus 1.9- to 19.4-fold at 5.0 mm). Furthermore, the stratum corneum

443

penetration contours of liposomes encapsulating NR versus liposomes encapsulating SRB are

444

distinct, unlike the ductal penetration contours (Figure S3). Therefore, the fluorescent dyes are

445

likely released from liposomes either at the tissue surface or within the 45-90 µm thick epidermis

446

33,

447

beyond the epidermis is likely a function of the penetration characteristics of the fluorescent

448

dyes themselves, rather than liposome-aided penetration. The conclusion that vesicles

449

destabilize at the tissue surface or after penetrating the tissue is further supported by the

as suggested by others 24, 34. The fluorescence intensity at extended penetration distances


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450

stability of the liposomes on the nipple surface during topical application for 6 hours (Figure S5).

451

There was no statistically significant change in liposome diameter before and after treatment.

452

Therefore, differences in the fluorescence intensity profiles between the two dyes are likely due

453

to release of encapsulated dyes upon liposome destabilization within the tissue, rather than at

454

the surface during topical application.

455

In contrast, the peak fluorescence intensity at the tissue surface likely is influenced by

456

liposome encapsulation. The peak fluorescence intensity at the stratum corneum surface was

457

reduced for liposomes containing the hydrophilic dye (SRB). Minimal differences were found

458

between the peak stratum corneum fluorescence intensities of NR encapsulating liposomes or

459

the NR solution (Figure 6A), suggesting that liposomes encapsulating NR and the NR solution

460

are equally capable of penetrating the stratum corneum. However, SRB administered via

461

liposomes had significantly decreased peak fluorescence intensities within the stratum corneum

462

compared to the SRB solution control (Figure 6B). At 1.0 mm in nipple depth, liposomes

463

encapsulating SRB had between 1.6- and 3-fold lower peak stratum corneum fluorescence

464

intensities as compared to the SRB solution. As nipple depth increased, the peak fluorescence

465

intensities gradually decreased, however the pronounced difference between liposomes

466

encapsulating SRB and the SRB solution was maintained. The overall reduction in peak

467

fluorescence intensity suggests that liposome encapsulation limited SRB penetration into the

468

stratum corneum from the donor solution.



469 470

Figure 6. Peak fluorescence intensities from NR (A) or SRB (B) penetration profiles of the

471

stratum corneum (n=3-6). Statistical analysis was performed with two-way ANOVA and Tukey’s

472

multiple comparison’s post-test. p ≤ 0.05 was considered significant and is denoted using the

473

following indicators: a- solution vs unextruded, b- solution vs 400 nm extruded, c- solution vs

474

100 nm extruded, d- unextruded vs 400 nm extruded, e- unextruded vs 100 nm extruded, f- 400

475

nm extruded vs 100 nm extruded.

476 477

Among the liposome formulations, a size effect was found such that the smallest

478

liposome (117.6 – 123.6 nm) had the highest peak stratum corneum fluorescence intensities,

479

while the largest liposome (2875 – 3340 nm) had the least (Figure 6A & 6B). These results are

480

consistent with the overall amount of dye penetrating the nipple (Figure 2) and with results from


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481

transdermal penetration 27, suggesting that dye penetration across the outer skin layers is

482

inversely related to liposome size.

483 484 485

CONCLUSION Drug delivery directly within the mammary ductal network offers an alternative treatment

486

strategy for pre-cancerous breast lesions that would maximize drug exposure within the

487

diseased tissue, while sparing healthy tissue. The results of the present study demonstrate the

488

feasibility of liposomes to skew penetration into and through the mammary ducts, while limiting

489

penetration via the stratum corneum. Liposomes, composed of EPC and ranging in size from

490

117 to 3,340 nm, resourcefully deposited both lipophilic NR and hydrophilic SRB into the

491

porcine nipple. Although total penetration of fluorescent dye into the nipple was reduced (SRB)

492

or marginally varied (NR) with liposome encapsulation, the fluorescence intensity, and therefore

493

dye concentration, at the ductal edge was increased up to 28-fold, suggesting deeper

494

penetration through the mammary ducts. Furthermore, the penetration distance into the

495

connective tissue via the ducts was reduced, indicating improved dye retention within the

496

mammary ducts. The consistent effect of liposome encapsulation on both the model hydrophilic

497

and lipophilic dye implies the vesicles remained intact during ductal diffusion; however for

498

transepidermal diffusion, consistency between the model fluorescent dyes was not found.

499

Liposomes made no impact on stratum corneum penetration for NR, but encapsulation

500

significantly reduced penetration for SRB. Beyond the stratum corneum, penetration profiles no

501

longer coincided for each dye, suggesting the dyes had been released from the liposome

502

vesicles.

503

While the current investigation focused on a single liposome composition and limited

504

size range, future studies will establish the impact of these important variables on transpapillary

505

delivery. Overall, the results indicate that liposomes may be promising vehicles for strategic

506

diffusion through the mammary ducts and future studies will further explore their transport in



507

vivo using fluorescent dyes and/or clinically relevant drugs to establish the therapeutic efficacy

508

of this localized administration technique.

509 510 511

ACKNOWLEDGEMENTS This work was supported by funding from the Tulane University School of Medicine and

512

a Tulane University Carol Lavin Bernick Faculty Grant. The authors gratefully acknowledge

513

Anita Verdun of Verdun Farm Meat Market for providing porcine tissue used in our investigation.

514 515

ABBREVIATIONS

516

EE, Encapsulation efficiency; EPC, Egg phosphatidylcholine; NR, Nile Red; PBS, Phosphate

517

buffered saline; PDI, Polydispersity index; ROI, regions of interest; SRB, Sulforhodamine B

518 519

SUPPORTING INFORMATION

520

Figure S1. Representative fluorescence montage micrographs of porcine nipple cross sections

521

following treatment with liposome-encapsulated nile red or nile red in solution

522

Figure S2. Representative fluorescence montage micrographs of porcine nipple cross sections

523

following permeation with liposome-encapsulated sulforhodamine B or sulforhodamine B in

524

solution.

525

Figure S3. Superimposed liposome fluorescence intensity curves of nipple tissue treated with

526

liposome-encapsulated nile red or sulforhodamine B.

527

Figure S4. Peak fluorescence intensity for each duct as a function of nipple depth.

528

Figure S5. Average liposome diameter before and after 6-hour topical application on a nipple.

529 530

REFERENCES


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induced dendritic cell maturation using a design of experiments approach. Eur J Pharm Biopharm 2015, 94, 427-35. 19. Mohammed, A. R.; Weston, N.; Coombes, A. G.; Fitzgerald, M.; Perrie, Y. Liposome formulation of poorly water soluble drugs: optimisation of drug loading and ESEM analysis of stability. Int J Pharm 2004, 285, (1-2), 23-34. 20. Clogston, J. D.; Patri, A. K. Zeta potential measurement. Methods Mol Biol 2011, 697, 63-70. 21. Smith, M. C.; Crist, R. M.; Clogston, J. D.; McNeil, S. E. Zeta potential: a case study of cationic, anionic, and neutral liposomes. Anal Bioanal Chem 2017, 409, (24), 5779-5787. 22. Gulati, M.; Grover, M.; Singh, S.; Singh, M. Lipophilic drug derivatives in liposomes. International Journal of Pharmaceutics 1998, 165, (2), 40. 23. Nii, T.; Ishii, F. Encapsulation efficiency of water-soluble and insoluble drugs in liposomes prepared by the microencapsulation vesicle method. Int J Pharm 2005, 298, (1), 198-205. 24. Peralta, M. F.; Guzmán, M. L.; Pérez, A. P.; Apezteguia, G. A.; Fórmica, M. L.; Romero, E. L.; Olivera, M. E.; Carrer, D. C. Liposomes can both enhance or reduce drugs penetration through the skin. Sci Rep 2018, 8, (1), 13253. 25. Rangsimawong, W.; Opanasopit, P.; Rojanarata, T.; Duangjit, S.; Ngawhirunpat, T. Skin Transport of Hydrophilic Compound-Loaded PEGylated Lipid Nanocarriers: Comparative Study of Liposomes, Niosomes, and Solid Lipid Nanoparticles. Biol Pharm Bull 2016, 39, (8), 1254-62. 26. Bhatia, A.; Kumar, R.; Katare, O. P. Tamoxifen in topical liposomes: development, characterization and in-vitro evaluation. J Pharm Pharm Sci 2004, 7, (2), 252-9. 27. Verma, D. D.; Verma, S.; Blume, G.; Fahr, A. Particle size of liposomes influences dermal delivery of substances into skin. Int J Pharm 2003, 258, (1-2), 141-51. 28. Krishnaiah, Y. S.; Bhaskar, P.; Satyanarayana, V. Penetration-enhancing effect of ethanol-water solvent system and ethanolic solution of carvone on transdermal permeability of nimodipine from HPMC gel across rat abdominal skin. Pharm Dev Technol 2004, 9, (1), 63-74. 29. Lademann, J.; Richter, H.; Teichmann, A.; Otberg, N.; Blume-Peytavi, U.; Luengo, J.; Weiss, B.; Schaefer, U. F.; Lehr, C. M.; Wepf, R.; Sterry, W. Nanoparticles--an efficient carrier for drug delivery into the hair follicles. Eur J Pharm Biopharm 2007, 66, (2), 159-64. 30. Lademann, J.; Weigmann, H.; Rickmeyer, C.; Barthelmes, H.; Schaefer, H.; Mueller, G.; Sterry, W. Penetration of titanium dioxide microparticles in a sunscreen formulation into the horny layer and the follicular orifice. Skin Pharmacol Appl Skin Physiol 1999, 12, (5), 247-56. 31. Toll, R.; Jacobi, U.; Richter, H.; Lademann, J.; Schaefer, H.; Blume-Peytavi, U. Penetration profile of microspheres in follicular targeting of terminal hair follicles. J Invest Dermatol 2004, 123, (1), 168-76. 32. Patzelt, A.; Richter, H.; Knorr, F.; Schäfer, U.; Lehr, C. M.; Dähne, L.; Sterry, W.; Lademann, J. Selective follicular targeting by modification of the particle sizes. J Control Release 2011, 150, (1), 45-8. 33. Kolliker, A., Manual of Human Histology. FB & C Limited: 2015; Vol. 1 (Classic Reprint).


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Figure 1. Determination of fluorescence intensity profiles using ImageJ Plot Profile tool. Sequential images of an entire porcine nipple cross section were compiled to create a single fluorescence micrograph (A). The line tool in ImageJ was then drawn across the stratum corneum or ductal lumen (B, blue line) to generate the fluorescence pixel intensity as a function of distance (C). For stratum corneum penetration, eight intensity profiles were averaged per tissue section. For ductal penetration, five intensity profiles were averaged per duct. 177x60mm (300 x 300 DPI)


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Figure 2. Dye retention in porcine nipple after in vitro diffusion for 6 hours. NR (A-B) or SRB (C-D) is reported as total percent applied (A,C) or delineated by depth (B,D) when applied in solution or encapsulated within liposomes. Data is represented as mean ± SEM (n=6). Statistical analysis was performed with one-way ANOVA (Retained Dye %) or two-way ANOVA (Retained Dye nmol/g) with Tukey’s multiple comparisons post-test. p ≤ 0.05 was considered significant and is denoted using the following indicators: a- solution vs unextruded, b- solution vs 400 nm extruded, c- solution vs 100 nm extruded, dunextruded vs 400 nm extruded, e- unextruded vs 100 nm extruded, f- 400 nm extruded vs 100 nm extruded. 140x125mm (300 x 300 DPI)



Figure 3. Representative fluorescence micrographs of mammary ducts and fluorescence intensity profiles for liposome- or solution-deposited dye. Porcine nipples were treated with NR (A) or SRB (B) either in solution or encapsulated within liposomes in vitro for 6 hours. Each curve is an average of 5 ROIs per duct (n=6-12). White scale bars in lower right corner of images represent 100 µm. 177x195mm (300 x 300 DPI)


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Figure 4. Peak fluorescence intensities from NR (A) or SRB (B) ductal penetration profiles (n=6-12). Statistical analysis was performed with two-way ANOVA and Tukey’s multiple comparison’s post-test. p ≤ 0.05 was considered significant and is denoted using the following indicators: a- solution vs unextruded, bsolution vs 400 nm extruded, c- solution vs 100 nm extruded, d- unextruded vs 400 nm extruded, eunextruded vs 100 nm extruded, f- 400 nm extruded vs 100 nm extruded. 84x115mm (300 x 300 DPI)



Figure 5. Representative fluorescence micrographs of the stratum corneum and fluorescence intensity profiles following in vitro diffusion of fluorescent dyes in solution or within liposomes. Porcine nipples were treated with NR (A) or SRB (B) either in solution or encapsulated within liposomes in vitro for 6 hours. Each curve is an average of 8 ROIs per tissue section (n=3-6). White scale bars in lower right corner of images represents 500 μm. 177x195mm (300 x 300 DPI)


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Figure 6. Peak fluorescence intensities from NR (A) or SRB (B) penetration profiles of the stratum corneum (n=3-6). Statistical analysis was performed with two-way ANOVA and Tukey’s multiple comparison’s posttest. p ≤ 0.05 was considered significant and is denoted using the following indicators: a- solution vs unextruded, b- solution vs 400 nm extruded, c- solution vs 100 nm extruded, d- unextruded vs 400 nm extruded, e- unextruded vs 100 nm extruded, f- 400 nm extruded vs 100 nm extruded. 84x115mm (300 x 300 DPI)


Liposomes Enhance Dye Localization within the Mammary Ducts of

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