LinearâDendritic Polymer Reverse

Subscriber access provided by University of South Dakota

Article

Evaluation of amphiphilic star/linear-dendritic polymer reverse micelles for transdermal drug delivery: directing carrier properties by tailoring core-vs-peripheral branching Karolina Anna Kosakowska, Brittany Kaylee Casey, Samantha Leigh Kurtz, Louise B Lawson, and Scott Michael Grayson Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00680 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 51 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

Biomacromolecules

Evaluation of amphiphilic star/linear-dendritic polymer reverse micelles for transdermal drug delivery: directing carrier properties by tailoring core-vs-peripheral branching Karolina A. Kosakowska †, §, Brittany K. Casey †, Samantha L. Kurtz ‡, §, Louise B. Lawson ‡, Scott M. Grayson †, * †

Department of Chemistry, School of Science and Engineering, Tulane University, New Orleans LA 70118 ‡

Department of Microbiology and Immunology, School of Medicine, Tulane University, New Orleans LA 70112 §

Bioinnovation PhD Program, School of Science and Engineering, Tulane University, New Orleans LA 70118 *Correspondence to S.M. Grayson (e-mail: [email protected])

KEYWORDS. Polymer architecture, branching location, amphiphilic self-assembly, reverse micelle, transdermal drug delivery, Franz diffusion cell, dye encapsulation

ACS Paragon Plus Environment

Biomacromolecules 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

ABSTRACT.

Page 2 of 51

The reverse micelle self-assembly of lipophile-functionalized poly(ethylene

glycol) (PEG) dendrimer hybrids is probed for applications in carrier-mediated transdermal drug delivery.

Under investigation are topologically diverse amphiphiles featuring controlled

branching motifs at either the polymer core (one-, two- and four-arm PEG) and/or the polar/nonpolar interface (peripheral dendritic generations 0 to 2). Thus, a systematic investigation of the effect of branching location (core versus peripheral) on carrier properties is described. Dyeencapsulation experiments verify these materials are capable of forming well-defined aggregates and solubilizing polar compounds. Further quantification of reverse micelle critical micelle concentration (CMC) and dye loading capacity for the branched amphiphile library was obtained through spectroscopy characterization.

Both core and peripheral branching are shown to

significantly influence dynamic encapsulation behavior, with evidence of location-based contributions extending beyond multiplicity of branching alone. Finally, the in vitro transdermal diffusion of the reverse micelle carriers was investigated through Franz diffusion cell experiments using physiologically relevant juvenile porcine dermis. The permeation results, combined with previously reported aggregate size trends, show the complex relationship between polymer branching and transdermal transport, with the lowest core- and highest peripherallybranched amphiphilic analog exhibiting optimal transdermal permeation characteristics for this set of branched carriers. INTRODUCTION Transdermal drug delivery offers a convenient, non-invasive route for parenteral drug administration that avoids the requisite trained personnel and adverse risks associated with parenteral injections.1,2 Likewise, this route circumvents the hepatic first-pass metabolism and harsh gastrointestinal conditions of orally administered (enteral) therapies, thereby improving


2

Page 3 of 51 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

Biomacromolecules

bioavailabilty.3

Yet, despite these advantages, fewer than two dozen drugs are currently

marketed as transdermal patches,4 predominantly due to the challenge of overcoming the skin’s innate barrier function.5, 6 Unassisted dermal permeation is believed to be limited to small (~500 Da),7 neutral8 molecules having a low melting point,9 solubility in both polar and non-polar media, a suitable octanol/water partition coefficient10 and high potency, thus requiring a relatively low effective dose.11 To bypass these restrictions, various approaches have been developed to facilitate transdermal drug delivery; these include chemical and biochemical permeation enhancers, dermal ablation, iontophoresis, sonophoresis,12 microneedle arrays13 and nanoparticle carriers.14-16

Although each offers unique advantages and limitations, a well-

designed nanoparticle vehicle may avoid the temporary disruption of the skin’s structural tissue components, which is a requirement of many of the other permeation enhancing techniques. Of the many physical, chemical and biological17 considerations in the design of transdermal carriers,18,19 the most crucial pertain to nanoparticle dimensions.20 The outermost layer of the epidermis—the stratum corneum (SC)—presents the greatest barrier in obstructing transdermal permeation as it comprises a tightly packed “brick and mortar” array of corneocytes surrounded by a lipid-rich extracellular matrix.21 To traverse the narrow intercellular lipid channels,22 carriers must be sufficiently small and/or deformable in shape, yet stable enough to diffuse intact without disrupting the tissue.23 Furthermore, maximizing the available cargo space for drug encapsulation is critical to ensure sufficient therapeutic bioavailability is maintained throughout the sustained diffusion process.24 Addressing these requirements, polymer-based nanoparticles present an attractive means for optimizing carrier behavior, as their physical properties can be readily tuned through modification of composition, structure and formulation.


3


Page 4 of 51

As such, amphiphilic copolymer aggregates have been extensively investigated for drug delivery application. Such materials typically exhibit greater stability, markedly lower critical micelle concentration (CMC), enhanced drug retention and smaller, narrower particle distributions when compared against small-molecule surfactant micelles or phospholipid-based liposomes.25 Several polymeric transdermal carrier systems have been previously investigated, employing a diverse range of materials and architectures. For example, poly(ethylene glycol)block-poly(D,L-lactic acid) (PEG-b-PLA) micelles were shown by Li et. al to enhance in vitro paclitaxel skin permeation roughly 5.5-fold as compared to drug alone, with evidence suggesting SC penetration occurs via a copolymer-induced route.26 Yang et. al investigated the effect of size and surface functionalization on in vitro transdermal diffusion of dye-conjugated poly(aminoamine) (PAMAM) dendrimers, wherein the smaller [G2] dendrimers exhibited 5.8times better epidermal absorption than the larger [G4] analogs. Moreover, partial oleic acid surface conjugation led to enhanced SC partitioning. The greatest permeation was observed with the acetylated “neutral” surface derivative, however.27 Previous studies from our group detail the transdermal carrier efficacy of dye-loaded reverse micelles prepared from amphiphilic 1-, 6and 12-arm star poly(oligo(ethylene glycol) methacrylate)-block-poly(lauryl methacrylate) (pOEGMA-b-pLMA).

In this case, increasing core branching multiplicity was shown to

enhance the magnitude and distance of in vitro dye-loaded carrier penetration.28 Conversely, Uhrich et. al report their unimolecular star amphiphilic carrier—featuring an acylated mucinic acid core and PEG corona—hindered percutaneous flux of various encapsulated non-steroidal anti-inflammatory drugs (NSAIDs), relative to the drug alone.29,30 In this later case, reduced transport may have been a consequence of carriers’ preorganized normal micellar structure, rather than the dynamic reverse micellar structure utilized in the present contribution. Overall,


4

Page 5 of 51 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

Biomacromolecules

the disparate outcomes reported for these assorted amphiphilic transdermal carrier systems indicate that polymer architecture can have a complex role in transdermal permeation which merits deeper investigation. Towards that end, this study aims to probe the structure-property relationships arising from polymer branching as applied to carrier-mediated transdermal drug delivery. Specifically, the effect of branching location—core versus peripheral—is investigated by assessing the selfassembly behavior across nine architecturally distinct amphiphiles. These materials, comprising lipophile-functionalized poly(ethylene glycol) (PEG) cores, feature a controlled number of branching motifs at the core of the PEG domain and/or at the polar/non-polar interface of the polymer periphery.

In non-polar media, self-assembly of these amphiphiles results in the

formation of reverse micelle aggregates, with a solvatophilic fatty acid corona surrounding an interior solvatophobic PEG microdomain.

A systematic comparison of reverse micelle

encapsulation capacity and CMC of the branched amphiphile library is explored and contextualized against the observed physical behavior of each of the polymer architectures to determine how branching location can affect dynamic aggregation properties. Furthermore, in vitro Franz cell dermal permeability was assessed, to probe the architecture-dependent behavior of the amphiphilic carriers during transdermal diffusion. EXPERIMENTAL Materials.

Branched amphiphiles were synthesized as reported elsewhere.31

Proflavine

hemisulfate salt (proflavine HS) was purchased from Millipore-Sigma (St. Louis, MO). 6Diamidino-2-phenylindole (DAPI), phosphate buffered saline (PBS), formalin (1:10 dilution, buffered), sucrose and all solvents were purchased from Fisher Scientific Inc. (Hampton, NH); materials were used without further purification.


5


Page 6 of 51

Reverse Micelle Preparation and Dye Encapsulation. Branched amphiphile aggregates were prepared in toluene from a known amount of dried polymer. The mixtures were repeatedly agitated (via vortex mixer) until fully dissolved and allowed to equilibrate overnight. To obtain a range of concentrations of amphiphile in toluene, serial dilution was used from a filtered stock solution (0.2 µm, Whatman, Maidstone, UK). For dye loaded carriers, the system was saturated with excess solid proflavine HS (typically, 0.5—1.0 mg mL-1 of amphiphile-toluene solution), placed on an orbital shaker for 2—4 h, and the mixture was left to settle at room temperature. As proflavine HS is extremely insoluble in toluene (< 3 uM, as determined in Supporting Information Fig. S1), the toluene supernatant was separated from undissolved dye and passed through a 0.2 µm syringe filter (Whatman) to remove any unencapsulated proflavine HS from solution. Samples were allowed to equilibrate following filtration (at least 30 min) prior to subsequent data collection. Spectroscopy Characterization. Absorption and fluorescence spectroscopy was performed to characterize reverse micelle CMC and dye encapsulation capacity. Measurements were collected in replicate (n ≥ 3) over a range of amphiphile concentrations. UV-vis measurements were collected on a Hewlett-Packard 8452A diode array spectrophotometer (Hewlett-Packard Company, Palo Alto, CA) and recorded using Olis SpectralWorks software (Olis, Inc., Bogart, GA). Relative dye encapsulation per carrier species was approximated via the Beer-Lambert relationship using molar extinction coefficients derived from proflavine HS in water. Fluorescence spectra were recorded on a PTI-Felix Fluorimeter (Photon Technology International, HORIBA Scientific, Edison, NJ) over a 425—750 nm wavelength region using a 2 nm slit width and an excitation wavelength of 410 nm.


6

Page 7 of 51 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

Biomacromolecules

In Vitro Transdermal Penetration. Juvenile porcine dermal tissue (Stellen Medical, St. Paul, MN; shaved, non-perforated, split thickness at 1.5 mm) was stored at -20°C. Upon use, the tissue was cut into approximately 2 cm x 2 cm sections, thawed at room temperature, and mounted onto a jacketed Franz diffusion cell (surface area 0.64 cm2; PermeGear Inc., Hellertown, PA), such that the stratum corneum was facing the donor chamber. The receptor chamber, containing ~5 mL of PBS, was maintained at 37°C and gently stirred throughout the experiment. The donor chamber was filled with 200 µL of dye-loaded carriers solubilized in toluene for 90 min. Upon completion, the remaining dye-carrier solution was aspirated from the donor chamber and the tissue surface was gently patted dry. Carrier-exposed samples were fixed in 10% formalin for 24 h, followed by successive 20, 30, and 40% sucrose solutions for 24 h each before cryopreservation in optimum cutting temperature compound (Tissue-Tek O.C.T Compound, VWR International). Tissues were sectioned using a Leica CM1860 cryostat (7 µm thickness; Leica Biosystems, Inc., Wetzlar, Germany), mounted on slides, and stained with the nuclear stain DAPI prior to sealing and storage. Carrier-exposed tissue cross sections were imaged via fluorescence microscopy on a Nikon A1 Confocal Microscope System (Nikon Instruments Inc., Melville, NY) using argon laser excitation wavelengths (λex) of 405 and 488 nm (blue and green channels, respectively) set to a pervasive incident laser intensity and detector gain for all captures. independent cross-sections were imaged per sample under 20x objective.

A minimum of 3 ImageJ software

(1.49v, NIH, Bethesda, Maryland, USA) with Nikon ND2 Reader plugin (Nikon Instruments Inc.) was used for image analysis.32 For quantification of mean fluorescence signal, the green (λex = 488 nm) channel capture was converted to 8-bit grayscale and the intensity was measured per select regions of interest (ROI) in the image. Obtained intensities were background corrected


7


Page 8 of 51

and normalized as a fraction of the total range (255) of pixel values prior to graphing. Following imaging of treated tissue cross sections, the relative fluorescence intensity under 488 nm excitation was quantified per skin region—namely, the outermost stratum corneum (SC), the subjacent viable epidermis (VE) and the deeper underlying dermal (UD) tissue layer.

RESULTS AND DISCUSSION Amphiphile Architecture and Reverse Micelle Self-Assembly. Chemical structures for the investigated branched amphiphile library are depicted in Scheme 1, with core branching varying along the vertical axis and peripheral branching varying along the horizontal axis. Materials were obtained through a modular synthetic route (detailed elsewhere)31, allowing for controlled incorporation of the branched motifs. Monofunctional “1-arm” monohydroxy monomethyl ether PEG (C1), bisfunctional “2-arm” dihydroxy PEG (C2) and tetrafunctional “4-arm” pentaerythritol-initiated PEG tetraamine (C4) of approximately equivalent masses (Mn = 5,000 Da) served as starting materials to provide the range of core branching.

Subsequent

dendronization with polyester dendrons based on 2,2-bis(hydroxymethyl)propionic acid (bisMPA) (dendritic generations [G0] through [G2]) was used to provide peripheral branching variability at the polar/non-polar interface for each of the PEG cores. Finally, comprehensive lauric acid esterification of the peripheral alcohol groups on each of the linear-dendritic hybrids incorporated the requisite lipophilic domain, yielding nine architecturally-distinct amphiphilic macromolecules (Scheme 1).

Importantly, the described branched polymers are both

biocompatible and biodegradable,33,34 in keeping with the proposed transdermal drug delivery application. Characterization by gel permeation chromatography (GPC), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and 1H/13C nuclear


8

Page 9 of 51 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

Biomacromolecules

magnetic resonance (NMR) analysis confirmed these materials are exceptionally well-defined.31 Table 1 summarizes the relevant amphiphile characterization data, with additional information provided in Table S1 of the Supporting Information.

Structural and chemical purity was

maintained throughout all steps of amphiphile synthesis, giving access to a library of amphiphilic polymers with controlled branching both with respect to multiplicity and location.

Table 1. Molecular weight, dispersity, and polarity ratio characteristics of investigated branched amphiphiles, as obtained from GPC, MALDI and 1H NMR characterization. Species Core Periphery Mn a Ɖb φLE c C1-(PEG-LE)1

[G0]

5240

1.026

3.1

[G1]

5545

1.027

6.0

C1-(PEG-[G2]-LE4)1

[G2]

6130

1.027

10.9

C2-(PEG-LE)2

[G0]

5175

1.029

6.4

[G1]

5760

1.030

11.2

C2-(PEG-[G2]-LE4)2

[G2]

6960

1.029

18.7

C4-(PEG-LE)4

[G0]

5985

1.029

10.9

[G1]

7140

1.034

18.4

[G2]

9510

1.033

27.7

C1-(PEG-[G1]-LE2)1

C2-(PEG-[G1]-LE2)2

C4-(PEG-[G1]-LE2)4 C4-(PEG-[G2]-LE4)4

1-arm

2-arm

4-arm

a

obtained from MALDI-TOF MS; b obtained from GPC analysis; distributions quantified using PEG standards for calibration; c mass % lipophilic laurate ester content per total amphiphile mass, calculated from 1H NMR integration.


9


Page 10 of 51

Scheme 1. Chemical structures of the investigated branched amphiphiles, featuring control over the extent of branching at the core (vertical axis) and at the periphery (horizontal axis).

In non-polar media, these branched amphiphiles self-assemble to form reverse micelles, with a solvatophilic lipid corona surrounding a solvatophobic polar PEG core microenvironment.35 As is the case for aqueous micellization (i.e. polar shell, non-polar core), the polymer composition directs self-assembly behavior. The relevant structural parameters include monomer identity, chain length, amphiphile polarity ratio, architecture and solvent compatibility.36-38

In a

concurrent study investigating aggregate size and dispersity for this specific architectural library of amphiphiles, it was found that branching multiplicity had a stronger effect than branching location in controlling self-assembly behavior (detailed elsewhere).31 Increased amphiphile branching (at either the core or periphery) was seen to decrease reverse micelle size and dispersity; results obtained by dynamic light scattering (DLS) are included in Table 2. In the


10

Page 11 of 51 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

Biomacromolecules

present study, the CMC, encapsulation capacity, and the in vitro transdermal permeability of each of the branched amphiphile carriers was determined and evaluated relative to branching location and multiplicity.

Table 2. Branched amphiphile reverse micelle physical properties, including aggregate size and dispersity, CMC (by fluorescence and UV absorbance spectroscopy) and dye encapsulation capacity. Diameter a PDI a

Species (nm)

λMax,Em b

CMCFl.

CMCAbs.

PF / Polymer c

(nm)

(mM)

(mM)

(mg g-1)

C1-(PEG-LE)1

161 ± 24

0.43 ± 0.05

488.0

0.85

0.68

0.02 ± 0.01

C1-(PEG-[G1]-LE2)1

137 ± 17

0.41 ± 0.04

492.5

0.45

0.41

0.08 ± 0.01

C1-(PEG-[G2]-LE4)1

64 ± 6

0.32 ± 0.03

502.3

0.26

0.24

0.32 ± 0.03

103 ± 13

0.38 ± 0.04

510.3

0.41

0.40

1.19 ± 0.05

C2-(PEG-[G1]-LE2)2

73 ± 8

0.34 ± 0.03

504.5

0.13

0.17

0.26 ± 0.04

C2-(PEG-[G2]-LE4)2

28 ± 3

0.20 ± 0.02

498.5

0.08

0.08

0.27 ± 0.02

C4-(PEG-LE)4

70 ± 9

0.33 ± 0.05

511.0

0.31

0.27

2.05 ± 0.14

C4-(PEG-[G1]-LE2)4

33 ± 3

0.20 ± 0.02

507.5

0.07

0.08

0.24 ± 0.02

C4-(PEG-[G2]-LE4)4

18 ± 2

0.15 ± 0.02

501.3

0.05

0.04

0.13 ± 0.01

C2-(PEG-LE)2

a

obtained by DLS intensity weighted distributions, experimental procedure detailed elsewhere31; b λMax values by fluorescence spectroscopy, reported for equivalent (~1 mM) amphiphile concentration; c weight ratio of encapsulated dye (proflavine HS) per amphiphile, calculated from UV-vis AMax values; data is presented as mean ± standard deviation. Effect of Branching Location on Amphiphile Reverse Micelle CMC.

Spectroscopy-

monitored encapsulation measurements were performed to evaluate the effect of polymer branching on aggregate CMC, employing the hydrophilic dye, proflavine hemisulfate salt (proflavine HS), as the fluorescent probe. Due to the insolubility of proflavine HS in nonpolar


11


Page 12 of 51

solvent, undissolved dye can be readily removed from a toluene solution of the polymeric reverse micelles via filtration, such that only dye internalized within the aggregate’s polar core microenvironment remains.39

A visible coloration was observed in dye-saturated toluene

solutions of the branched amphiphiles as compared to relevant control studies (Supporting Information, Fig. S2 and S3), verifying the amphiphilic polymers are capable of aggregation and can effectively solubilize polar compounds (such as proflavine HS) in non-polar solvent. However, at concentrations below a species’ CMC, no apparent spectroscopic UV signal could be detected, as low amphiphile availability in solution favors its unimer state (rather than reverse micelle formation), hence the dye remains unsolubilized.36


12

Page 13 of 51 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

Biomacromolecules

Figure 1. Summary fluorescence spectroscopy data for proflavine HS-saturated branched amphiphile solutions, plotting dye intensity maxima (IMaxx10-5, a.u. with standard deviation, upper panel) and wavelength maxima (λMax, nm, lower panel) per polymer concentration (log mM), obtained and averaged over multiple runs (n ≥ 3); (a) C1-(PEG-LE)1; (b) C1-(PEG-[G1]LE2)1; (c) C1-(PEG-[G2]-LE4)1; (d) C2-(PEG-LE)2; (e) C2-(PEG-[G1]-LE2)2; (f) C2-(PEG[G2]-LE4)2; (g) C4-(PEG-LE)4; (h) C4-(PEG-[G1]-LE2)4; (i) C4-(PEG-[G2]-LE4)4. All axes are held constant, except for the intensity plots shown in 1c, 1d and 1g (upper panels), which have been scaled to include higher IMax values; a dashed line at 2.4x105 a.u. denotes the IMax range (i.e. maximum y-axis limit) for the remaining 6 amphiphile fluorescence graphs.


13


Page 14 of 51

The self-assembly of branched amphiphiles was assessed using fluorescence spectroscopy over a range of dye-saturated carrier concentrations (Fig. 1, Supporting Information Fig. S4). Emission properties of fluorescent probes are significantly influenced by the surrounding environment, allowing a robust assessment of dye internalization;40 thus, fluorescence monitoring of maximum proflavine HS intensity (IMax, Fig. 1, top panels) and wavelength (λMax, Fig. 1, bottom panels) provides complementary indications of the aggregate CMC’s. At very low polymer concentration (i.e. 10-fold below CMC), fluorescence spectra resemble the control studies obtained for amphiphile-toluene solutions without any dye (λMax = 464—470 nm, Fig. S5a), indicating no detectable proflavine HS retention because of the absence of stable reverse micelles at sub-aggregation dilutions.

As amphiphile concentration is increased, the

fluorescence maxima shift to longer wavelength (typically 474—486 nm, Fig. S5b and S5c) and a nominal increase in intensity is observed at the onset of the amphiphile-assisted dye solubilization. For this transient CMC concentration regime, the low measured Imax and λMax values suggest the dye is only modestly solubilized in an environment (i.e. the reverse micelle core) slightly more polar than the bulk toluene solvent. Finally, once above a species’ CMC, a substantial increase in signal intensity can be seen (Fig. S5d), with corresponding λMax shifting to progressively longer wavelengths, closer to that of the expected fluorescence spectra of proflavine HS in water (λMax = 510 nm, Supporting Information, Fig. S6) or a similar polar environment.41 The subtle bathochromic shift can be attributed to dye internalization within the reverse micelles’ PEG core, reflecting an increase in the polarity of the dye’s environment,42 as well as excimer interactions between multiple encapsulated proflavine HS molecules.43 Branched amphiphile CMC values were obtained as the intersect of fluorescence response trend lines with respect to amphiphile concentration fitted below and above the inflection point (Supporting


14

Page 15 of 51 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

Biomacromolecules

Information, Fig. S7)44 and are reported in Table 2. Both spectroscopy techniques provided analogous results, however due to the enhanced sensitivity of the fluorescence technique, these data will be primarily considered in following discussion of reverse micelle CMC behavior.

Figure 2. Branched amphiphile CMC (mM, log scale) values as obtained by fluorescence (solid line) and UV absorbance (dashed line) spectroscopy; (a) by core branching (x-axis); (b) by peripheral branching (x-axis); (c) 3-D bar graph of CMC (mM, via fluorescence) against both core (y-axis) and peripheral (x-axis) branching.


15


Page 16 of 51

The effect of branching multiplicity and location on amphiphile CMC was investigated to evaluate the role of polymer architecture in reverse micelle self-assembly. The obtained CMC values for the branched amphiphile library are plotted as a function of core and peripheral branching in Figure 2a and 2b, respectively (log mM scale).

At either location (core or

periphery), an increase in branching multiplicity decreases reverse micelle CMC, most likely in correlation with an increase in the overall lipophilic mass fraction (φLE) provided in Table 1. The importance of copolymer “amphiphilicity” (i.e. relative polar/non-polar mass ratio) on aqueous self-assembly behavior has been extensively investigated for classical micellization of linear block copolymer systems, and is known to significantly alter CMC properties, particularly in regard to the size of the micelle core-forming (non-polar) domain.45-47 Though less studied, similar correlations have been made for reverse micelle systems.48-50 For the amphiphile library investigated here, the mass of the core-forming PEG domain is constant for all branched analogs (Mn = 5,000 Da). Therefore, the differences in self-assembly CMC observed between species are solely a consequence of varying polymer architecture and alkyl content, two parameters which are inherently correlated. An increase in branching multiplicity at either location doubles the amount of laurate ester functionalities and affords a correspondent increase in the mass fraction of amphiphile lipophilic content. As CMC fundamentally relates to aggregate stability (the lower the CMC, the more thermodynamically stable the aggregate),51 branched amphiphiles with a larger solvatophilic mass fraction can provide greater reverse micelle coronal coverage and improved aggregate stability. Therefore, increased branching multiplicity at either the core or periphery may effectively stabilize the reverse micelle and afford lower aggregate CMC values, and appears to have a predominant effect on CMC behavior (i.e. CMC decreases with increased branching, irrespective of location), as seen by the analogous trends in Figure 2.


16

Page 17 of 51 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

Biomacromolecules

Assessing peripheral branching across the 1-arm PEG analogs (Fig. 2b, light gray line), a steady decrease in reverse micelle CMC in toluene is observed—roughly 1.8-fold—upon each additional generation (from [G0] to [G1] and [G1] to [G2]). This effect is consistent with other reports investigating the micellization of linear-dendritic amphiphiles in water. For examples, an increase in dendritic generation of polyglycidol-dendronized alkyl chains (C16-dend-PG)52 or of PLA-dend-PAMAM53 prompted a 1.5- and 2.2-fold decrease (by mM, per generation) in aggregate CMC, respectively. Interestingly, for the self-assembly of the 2- and 4-arm analogs, [G1] dendronization affected a greater a reduction in CMC (3.2- and 4.3-fold, respectively, when compared to the analogous [G0] amphiphile species) than with the one-arm analog. However, further dendronization (from [G1] to [G2]) saw a smaller decrease (approximately 1.6-fold) in reverse micelle CMC for each of the three cores (Fig. 2b, dark gray and black series). Therefore, even though increasing peripheral branching uniformly doubles amphiphile alkyl content, the effect this has on reverse micelle CMC is disproportionate between dendritic generations. Similar deviations in CMC trends were observed when evaluating the effect of core branching on branched amphiphile self-assembly behavior. For the [G0] dendronized amphiphiles, (Fig. 2a, light gray line), increasing branching multiplicity from 1- to 2- to 4-arms (C1-(PEG-LE)1, C2-(PEG-LE)2, and C4-(PEG-LE)4) decreases aggregate CMC from 0.85 to 0.41 to 0.31 mM, respectively. Here, the CMC decreases 2.1-fold upon increasing core branching from the 1-arm to 2-arm, but only 1.3-fold going from the 2-arm to the 4-arm PEG analogs.

A similar

asymmetry between core multiplicity increments was observed for both the [G1] (Fig. 2a, dark gray line) and [G2] (Fig. 2a, black line) peripherally dendronized amphiphiles. For either series, increasing branching from the C1 to C2 core decreases the reverse micelle CMC approximately twice as much as does increasing branching from C2 to C4 PEG core. Therefore, just as


17


Page 18 of 51

peripheral branching was seen to disproportionately modulate aggregate CMC, the same disproportionate behavior is observed between variations of core branching multiplicity. Taken together, these observed deviations in CMC trends indicate that, while total branching multiplicity is the predominate factor directing self-assembly behavior54, polymer architecture does indeed impact CMC as well. To probe the effects of branching location on reverse micelle CMC, a comparison between branched amphiphiles of equivalent branching and lipophile content (i.e. same number of lauryl groups) is depicted in Figure 3. First, addressing the selfassembly behavior in the tetra-laurylated species (Fig. 3b, top panel), the amphiphile with the highest degree of core branching (C4-(PEG-LE)4, 0.31 mM) has the highest observed CMC. Conversely, the lowest CMC in this amphiphile subset is seen for the bis-[G1]-branched species C2-(PEG-[G1]-LE2)2 (0.13 mM), with the 1-arm [G2] dendronized C1-(PEG-[G2]-LE2)1 (0.26 mM) as intermediate in value. Thus, despite each of these three amphiphiles having the same amount of branching and the same non-polar content (4 laurate groups each), the amphiphile with modest branching at both the core and periphery (C2-(PEG-[G1]-LE2)2) has the lowest reverse micelle CMC, approximately half that (2.2-fold lower) of either the entirely corebranched (4-arm) or entirely peripherally-branched ([G2]) tetra-laurylated analog.


18

Page 19 of 51 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

Biomacromolecules

Figure 3. Branched amphiphile reverse micelle CMC values (mM, top plot) and comparison of spectroscopy measured proflavine HS intensity per polymer concentration (I mM-1, values normalized relative to C4-(PEG-LE)4 intensities, bottom plot) as observed by fluorescence (dark fill) and UV absorption (light fill), grouped by equivalent total branching number/amphiphile polarity ratio; (a) 2 laurate ester functionalities; (b) 4 laurate ester functionalities; (c) 8 laurate ester functionalities.

When placed in context of the free energy theory of self-assembly,55 it appears that a high number of core- or peripherally-localized branching junctions imposes a greater conformational entropic penalty of aggregation, prompting an increase in CMC.

As such, architectural

incorporation of [G1] peripheral or C2 core branching was seen to decrease CMC to a larger extent than either [G2] or C4 branching, respectively, and the analog with more distributed branching can be expected to have a lower CMC. In the case of the octa-laurylated amphiphiles (Fig. 3c), reverse micelle CMC was lower for the 4-arm analog with four small [G1] dendrons (C4-(PEG-[G1]-LE2)4) than for the 2-arm analog with half as many large [G2] dendrons C2(PEG-[G2]-LE4)2). Similar results can be seen with the bis-laurylated species (Fig. 3a), where the 2-arm amphiphile (C2-(PEG-LE)2) with no dendrons exhibiting reduced aggregate CMC relative to a single [G1] dendron with a 1-arm core (C1-(PEG-[G1]-LE2)1). Future studies investigating self-assembly in 8-arm core- or [G3] peripherally-branched amphiphiles may help elucidate the generality of the observed CMC trends, namely a preference towards more distributed branching to achieve low CMCs.


19


Page 20 of 51

Effect of Branching Location on Amphiphile Reverse Micelle Dye Encapsulation. In addition to aggregate CMC determination, the amount of dye encapsulated within the reverse micelles was approximated through absorbance measurements using the Beer-Lambert relationship. A bathochromic shift of proflavine HS maximum absorbance from 442—446 nm (in H2O, Supporting Information, Fig. S8a) to 450—456 nm is observed and can be attributed to dye localization within the core reverse micelle microenvironment.39 For each carrier species, relative proflavine HS encapsulation was determined by UV-vis absorbance over a range of concentrations and reported as a weight ratio (mg g-1) of internalized dye mass-per-polymer mass in solution (Table 2). A summary of absorbance data and representative UV-vis spectra are depicted for each amphiphile in Figure S9 (Supporting Information). Complementary trends relating apparent dye intensity per amphiphile concentration were also observed by fluorescence spectroscopy (Fig. 3, bottom panels), however these were not relied upon for quantification because of the greater propensity for fluorescence quenching when utilizing this technique (Fig. S6).56 As illustrated by Figure 4, both peripheral and core branching play a significant role in carrier proflavine HS uptake, with trends deviating significantly from the predominant dependence on overall branching multiplicity observed for self-assembly CMC behavior.


20

Page 21 of 51 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

Biomacromolecules

Figure 4. Summary of branched amphiphile library dye encapsulation results based on UV-vis absorption data, plotted with standard deviation as the mass ratio (wt./wt., mg g-1) of encapsulated proflavine HS mass per polymer mass; (a) by core branching (x-axis); (b) by peripheral branching (x-axis); and (c) as a bubble plot with the circle area depicting relative dye encapsulation as a function of both core (y-axis) and peripheral (x-axis) branching.

Of the nine architecturally distinct polymers investigated, the 4-arm and 2-arm core species with no peripheral branching (C4-(PEG-LE)4 and C2-(PEG-LE)2) exhibited substantially higher wt./wt. aggregate proflavine HS loading than all other branched amphiphiles. Hence, a reduction of peripheral branching multiplicity at the amphiphilie solvatophobic/solvatophilic interface is a critical parameter for improving encapsulation capacity. For example, increasing generation number of the 4-arm derivatives (Fig. 4b, black line and 4c, bottom row) results in a stark decrease in aggregate encapsulation capacity. Proflavine HS uptake into these reverse micelles decreases from 2.05 mg g-1, to 0.24 mg g-1 and finally 0.13 mg g-1 for C4-(PEG-LE)4, C4-(PEG-[G1]-LE2)4, and C4-(PEG-[G2]-LE4)4, respectively. The same peripheral branching effect is observed when comparing 2-arm core amphiphiles (Fig. 4b, dark gray line and 4c,


21


Page 22 of 51

middle row), with the lowest degree of peripheral branching (C2-(PEG-LE)2) showing the highest measured dye intensity of the three analogs with a bis-functional core. This trend may likely be correlated to aggregate size (Table 2); peripheral branching decreases reverse micelle diameter, resulting in a greater corona-interfacing surface area per reverse micelle core volume. Hence, for smaller aggregates, a lower proportion of the polar core microenvironment is available for proflavine HS encapsulation.57 However, the opposite trend is seen for the 1-arm derivatives (Fig. 4b, gray line and 4c, top row), whereby increasing peripheral branching from C1-(PEG-LE)1 to C1-(PEG-[G1]-LE2)1 to C1-(PEG-[G2]-LE4)1 yields an increase in aggregate encapsulation capacity (0.02 mg g-1 to 0.08 mg g-1 to 0.32 mg g-1, respectively). This reversal may arise from the φLE of the less-branched 1-arm amphiphile species (i.e. C1-(PEGLE)1 and C1-(PEG-[G1]-LE2)1, Table 1), which have the lowest ratio of solvatophilic coronaforming domains relative to solvatophobic PEG arm length. As such, increased C1 peripheral branching is seen to improve dye solubilization, as the increased φLE may provide greater coronal shielding of the reverse micelle core microenvironment.58,59 Overall, the impact of peripheral branching on reverse micelle proflavine HS loading appears to be very dependent on the core used. Additional branching trends could be observed when viewing this data set with respect to core branching.

Comparing the aggregate encapsulation across amphiphiles with branching

exclusively at the core (i.e. [G0] species, Fig. 4a, gray line and 4c, left column), increased core branching is seen to enhance proflavine HS uptake. The 4-arm C4-(PEG-LE)4 exhibiting the greatest loading capacity of all branched species (2.05 mg g-1), with a dramatic increase in dye uptake as compared to the unbranched C1-(PEG-LE)1 (0.02 mg g-1). The trend of higher dye encapsulation with increased core branching is maintained for the [G1] dendronized amphiphile


22

Page 23 of 51 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

Biomacromolecules

series as well (Fig. 4a, dark gray line and 4c, center column), however at a substantially lower magnitude (0.08 – 0.26 mg g-1) as compared to the [G0] analogs. Similar results have been shown comparing reverse micelle dye encapsulation of alkylated 4-, 5- and 6-arm star poly(glycidol methacrylate) amphiphiles, where a higher degree of core branching resulted in greater aggregate uptake capacity (6-arm > 5-arm > 4-arm).60 One possible interpretation for this trend is that the increased number of solvatophobic PEG arms yields a more polar microenvironment in the reverse micelle core.

This hypothesis is further supported by

fluorescence-measured λmax values for the [G0] and [G1] series of PEG-dendrimer hybrids (Table 2). The increasing λmax with respect to increasing core multiplicity suggest a more polar core microenvironment; conversely, when λmax is low (such as the case for C1-(PEG-LE)1 and C1-(PEG-[G1]-LE2)1, Table 2), it may be expected that the poorly shielded reverse micelle cores cannot effectively encapsulate and solubilize proflavine HS.61

However, the core

branching trend observed for [G0] and [G1] is inverted for the [G2] analogs (Fig. 4a, black line and 4c, right column), with increasing core branching from C1-(PEG-[G2]-LE4)1 to C2-(PEG[G2]-LE4)2 to C4-(PEG-[G2]-LE4)4 yielding decreased proflavine HS encapsulation capacity (from 0.32 mg g-1 to 0.27 mg g-1 to 0.13 mg g-1, respectively).


23


Page 24 of 51

Figure 5. Proflavine HS encapsulation capacity of branched amphiphile reverse micelles determined by UV-vis measurement (n ≥ 3), plotted as a weight ratio of dye per polymer (wt./wt., mg g-1) and grouped by equivalent amphiphile polarity ratio/total number of peripheral groups; (a) 2 laurate ester functionalities; (b) 4 laurate ester functionalities; (c) 8 laurate ester functionalities.

Taken together, these results indicate that both core and peripheral branching multiplicity have an intricate—though generally opposing—effect on dye encapsulation capacity. Evaluating proflavine HS capacity between amphiphiles of equivalent total branching multiplicity (and, therefore, equivalent polarity ratio) offers further insight into branching location trends, as reverse micelle size, dispersity and CMC remain relatively consistent between equally branched species. As seen in Figure 5a and 5b for the bis- and tetra-laurylated analogs, the amphiphiles of higher core (and lower peripheral) branching exhibit significantly greater proflavine HS encapsulation. Conversely, for the highly-branched octa-laurylated amphiphiles (Fig. 5c), dye encapsulation is more equivalent—albeit moderately higher for C2-(PEG-[G2]-LE4)2 (0.27 mg g-1) than for C4-(PEG-[G1]-LE2)4 (0.24 mg g-1)—hence, branching location has less of an effect on encapsulation behavior at high branching multiplicity. Overall, the effect of branching location on reverse micelle proflavine HS uptake for the investigated library of amphiphiles yielded consistent—yet incongruent—trends in observed encapsulation behavior, indicating a complex interplay of contributing factors. Future studies of branched amphiphile self-assembly holding alternate polymer design parameters constant (i.e., alkyl content, PEG arm length, etc.) may serve to elucidate distinct contributions to the observed encapsulation properties. Nonetheless, though the exact origins of these trends remain unclear, it is apparent that polymer architecture does indeed play a significant role in defining the encapsulation behavior of reverse micelles.


24

Page 25 of 51 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

Biomacromolecules

In-Vitro Skin Permeability Investigation: Effect of Core and Peripheral Branching on Transdermal Permeation.

Finally, permeation of the branched amphiphile carriers was

assessed by in vitro transdermal diffusion studies to probe the effectiveness of each species in transporting dye across skin. In keeping with the ultimate application of transdermal drug delivery, juvenile porcine dermal tissue was used, as it has comparable properties to human skin.62 Polar drugs or compounds—such as proflavine HS—cannot penetrate (i.e. diffuse into) the skins outermost, highly lipophilic stratum corneum (SC) unassisted. Conversely, while nonpolar species can partition into the SC, these are expected to be retained on the skin’s surface, and cannot permeate (i.e. diffuse through) further into the hydrophilic viable epidermis (VE) and underlying dermis (UD) layers. Hence, for transdermal drug delivery, encapsulation within a lipid-soluble reverse micelle carrier could assist polar drug SC transport, allowing for triggered payload release and systemic uptake upon encountering the hydrophilic tissue below. Using a Franz diffusion cell (diagramed in the Supporting Information, Fig. S10), both the relative transport efficacy (i.e. intensity of dye penetration) and depth of transdermal diffusion (i.e. extent of dye permeation) into porcine skin was evaluated for each of the branched amphiphile carriers. To serve as negative controls, skin samples were treated with toluene only, proflavine HSsaturated toluene solution (no carrier), and proflavine HS in water (no carrier); the results are reported in Figure. S11 (S11a, S11b and S11c, respectively) and Table S2 in the Supporting Information. Following exposure to toluene alone (Fig. S11a), only minimal signal was detected in the green wavelength (λex = 488 nm) at every tissue depth and this limited background signal could be attributed to the skin’s native autofluorescence.63 Similar results were observed in samples treated with proflavine HS-saturated toluene solution (Fig. S11b), confirming no measurable dermal penetration occurred as proflavine HS is insoluble in the non-polar media and


25


Page 26 of 51

SC lipids. Importantly, tissue sections showed no major perturbations in dermal structure or skin integrity over the duration of toluene exposure (90 min), in agreement with previously reported studies.64

Furthermore, samples treated with proflavine HS in water showed strong dye

fluorescence, but this was confined to the SC with no change in underlying VE and UD signal intensity (Fig. S11c).

Therefore, despite the dye’s excellent aqueous solubility, the polar

formulation cannot bypass the lipophilic SC barrier, prompting negligible dye permeation beyond the outermost tissue surface. Additional control experiments were conducted using saturated proflavine HS solutions of discrete amphiphile components, namely PEG (Mn = 5,000 Da) and lauric acid, as well as a 6.3:1 (wt.:wt.) PEG:lauric acid mixture (this ratio of unconjugated components crudely mimics the composition of the tetra-laurylated amphiphiles), prepared in toluene.

In all cases, dye localization was limited to the outmost SC and no

measurable permeation was observed in the underlying tissue (Supporting Information, Fig. S12, Table S3). Thus, for the experimental conditions employed here, proflavine HS cannot diffuse across the SC exclusively with water or toluene as solvents. Likewise, controls with PEG and lauric acid alone confirm that—in the case that the carriers degrade—the branched amphiphile components themselves do not exhibit an appreciable improvement in the transdermal transport of proflavine HS.

Therefore, the observed increase in fluorescence intensity of the deeper tissue layers

following skin treatment with dye-loaded amphiphilic carriers (Fig. 6) can be attributed to carrier-mediated proflavine HS transport.


26

Page 27 of 51 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

Biomacromolecules

Figure 6. Representative fluorescence microscopy images of porcine dermis cross-sections (SC up) following 90 min exposure to 5.0 mg mL-1 proflavine HS-loaded carrier solution of (a) C1(PEG-LE)1; (b) C1-(PEG-[G1]-LE2)1; (c) C1-(PEG-[G2]-LE4)1; (d) C2-(PEG-LE)2; (e) C2(PEG-[G1]-LE2)2; (f) C2-(PEG-[G2]-LE4)2; (g) C4-(PEG-LE)4; (h) C4-(PEG-[G1]-LE2)4; (i) C4-(PEG-[G2]-LE4)4; right-hand panel shows green channel (λex = 488 nm) capture only; lefthand panel shows overlay of green and blue (λex = 405 nm) channels (nuclei stained blue with DAPI).


27


Page 28 of 51

Figure 7. Background corrected fluorescence intensity measured in the SC (top), VE (middle) and UD (bottom) of carrier-treated porcine dermis, plotted by (a) core branching; (b) peripheral branching. Values were averaged from multiple tissue sections (n > 3) and corrected for autofluorescence.

In accordance with CMC and dye encapsulation studies, in vitro experiments were performed using reverse micelles in toluene with proflavine HS as the fluorescent probe, to allow for a direct comparison between the various branched amphiphile data sets (including aggregate size determination, reported elsewhere).31 Hence, although toluene is a poorly suited vehicle for clinical transdermal application,65 it allows for preliminary investigation of carriers’ in vitro dermal transport to be evaluated and directly compared to the comprehensive physical characterization of the same aggregates in toluene. For each branched amphiphile species, carrier solutions of equivalent concentration (5.0 mg mL-1) were subject to dermal permeation studies, as well as at a secondary concentration (either 2.5 or 10.0 mg mL-1, depending upon aggregate signal intensity) to verify observed trends (Supporting Information, Figs. S13 and S14, Table S4). Even by visual evaluation of fluorescence intensity and location in tissue sections shown in Figure 6, polymer architecture exhibits a significant impact on carrier transdermal dye


28

Page 29 of 51 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

Biomacromolecules

transport. The observed green (λex = 488 nm) fluorescence per dermal region (SC, VE and UD) of carrier treated samples was quantified and corrected for tissue autofluorescence (obtained from control experiments, Table S2). The measured signal intensities prior to autofluorescence subtraction are reported in Figure S15 and Table S5 (Supporting Information). As seen in Figure 7, the relative loading capacity of proflavine HS per carrier is a defining parameter in the ultimate magnitude of fluorescence signal measured at each depth of the skin. As expected, the results plotted by core and peripheral branching resemble graphical dye encapsulation trends (Fig. 4a and 4b), particularly in the SC (Fig. 7, top panels). For example, aggregates with the highest encapsulation capacity—namely, C4-(PEG-LE)4 and C2-(PEG-LE)2—had the brightest measured SC signal (Fig. 6g and 6d, respectively). Therefore, to accurately assess the effect of branching on carrier-mediated dye diffusion independent of loading capacity, the relative intensity in each dermal region was plotted as the ratio of measured fluorescence signal per aggregate encapsulation capacity (i.e. measured signal intensity/carrier proflavine HS capacity in mg g-1, Fig. 8).


29


Page 30 of 51

Figure 8. Relative fluorescence intensity of carrier-treated tissue in the SC (top), VE (middle) and UD (bottom) adjusted per aggregate encapsulation capacity, plotted by (a) core branching; (b) peripheral branching. Assessing the relative intensity of carrier-mediated diffusion, core branching appears to be the key parameter for defining penetration into the outermost SC layer, whereas peripheral branching exhibits the strongest effect for permeation into the underlying VE (Fig. 8b and 8a, respectively). As seen in Figure 8b (top panel), the SC encapsulation-adjusted fluorescence values decrease with increased core branching for each generation of peripheral dendronization. This trend is most evident for the [G2] analogs and, while less pronounced, can still be distinguished when comparing core branching across [G0] carriers. In contrast, the effect of peripheral branching on relative intensity within the SC is varied. In the subjacent VE, however, the influence of peripheral branching is more significant (Fig. 8a, middle panel); in this underlying tissue region, the relative fluorescence signal increases for each subsequent generation of dendritic branching. Core branching effects yielded a similar trend within the VE—with increased branching enabling greater dye permeation—though to a lesser extent than peripheral branching. Furthermore, amphiphiles with greater peripheral branching exhibited greater proflavine HS localization within the UD when compared to analogs with a similar overall branching, but a larger percentage of that branching located near the core (Supporting Information, Fig. S16). For example, when comparing the bis-laurylated carriers, C1-(PEG[G1]-LE2)1 has a greater UD signal than C2-(PEG-LE)2. Similarly, for the tetra-laurylated cores, C1-(PEG-[G2]-LE4)1 exhibits greater signal than C2-(PEG-[G1]-LE2)2 which, in turn, was higher than with the C4-(PEG-LE)4 carrier. Finally, for the octa-laurylated cores, C2(PEG-[G2]-LE4)2 showed enhanced proflavine HS diffusion into the UD as compared to C4(PEG-[G1]-LE2)4. Overall, the results suggest that core branching most profoundly effects dye


30

Page 31 of 51 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

Biomacromolecules

penetration into the SC while peripheral branching influences depth of permeation. Furthermore, this observation can be better understood when considered in the context of aggregate size.

Figure 9. Effect of aggregate size on relative fluorescence intensity of carrier-treated tissue in the SC (top), VE (middle) and UD (bottom) adjusted per aggregate encapsulation capacity, plotted as a function of diameter (nm, x-axis) in series of (a) peripheral branching; (b) core branching; (c) all branched amphiphile carriers, distinguishing between peripheral (red, yellow, blue) and core (no pattern, sparse pattern, dense pattern) variability.

Efficacy of carrier-mediated dermal penetration was probed with respect to reverse micelle size, depicted in Figure 9 in series with respect to peripheral (Fig. 9a) or core (Fig. 9b) branching as a function diameter (nm). Within the SC, all carriers over 70 nm had mid-range encapsulation-adjusted fluorescence values; however, within the VE, these exhibited the lowest observed signal (Fig. 9c).

Thus, while the largest aggregates can successfully transport


31


Page 32 of 51

proflavine HS into the SC, further permeation into the underlying layers of the skin appears to be inhibited. Conversely, below 40 nm in diameter, carriers showed low SC dye retention but high VE permeation, with the smallest species—C4-(PEG-[G2]-LE4)4—having the highest VE signal. This data suggests that the smaller aggregates are more successful at diffusing through the outermost skin layer and permeating to the deeper tissue. Contextualized to branching trends in Figure 9a, increased peripheral branching corresponds to a progressive decrease in carrier size, such that the [G2] analogs (Fig. 9a, black fill) have the highest observed VE and UD signal. Likewise, proflavine HS intensities in the SC are greatest for the 1-arm derivatives (Fig. 9b, gray fill) and decrease upon increased core multiplicity, in coordination with decreasing reverse micelle diameter. However, with no verification of the stability of dye-loaded aggregates upon permeation into or through the skin, the precise mechanism of branched carrier transdermal transport is unknown. For example, dye-loaded carriers may permeate intact into the deeper skin layers to some extent, or may enhance permeation by promoting payload release in the SC.66,67 Nonetheless, as all dye-saturated control studies demonstrate no appreciable improvement of VE or UD intensity (Supporting Information, Table S2 and S3 and Fig. S11 and S12), the increased proflavine HS penetration observed with the carriers supports a carrier-mediated mechanism— and the importance of carrier size—for dye transport into and through the skin.68-70 Moreover, it is important to reiterate that while these trends confirm smaller carriers promote more effective skin permeation, their payload is also small, due to the lower encapsulation capacity. Therefore, in defining goals for future carrier design, an ideal system would be able to accommodate a large payload while simultaneously maintaining appropriately small dimensions. In summation, carrier efficacy in transdermal transport was predominantly influenced by reverse micelle size and dye-encapsulation capacity. Aggregates with low peripheral branching


32

Page 33 of 51 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

Biomacromolecules

and high proflavine HS loading capacity showed strong SC intensity, however penetration was generally restricted to the surface tissue.

Conversely, increasing either core or peripheral

multiplicity decreased aggregate size and led to enhanced dye permeation into the deeper tissue regions.

As a final note, C1-(PEG-[G2]-LE4)1 (64 nm in diameter) has both the highest

measured and encapsulation-adjusted fluorescence intensity within the UD, despite exhibiting a relatively low encapsulation capacity. This mid-sized carrier, with minimal core branching and high peripheral branching, highlights the complexity of physicochemical factors—beyond just size or encapsulation capacity—affecting penetration into and permeation across the skin.

CONCLUSIONS The reverse micelle self-assembly behavior was probed across an architecturally diverse library of amphiphilic polymers through spectroscopy-monitored dye encapsulation studies. The amphiphiles feature structural control over branching multiplicity at the polymer core (1-, 2- and 4-arm) as well as the periphery ([G0], [G1], and [G2] dendronization), allowing aggregate CMC and proflavine HS uptake capacity to be assessed as a function of branching location. Here, CMC trends were most dependent on total amphiphile branching number and corresponding overall polar/non-polar ratio, with lower values seen for amphiphiles of higher total branching/non-polar ratio.

Conversely, proflavine HS encapsulation capacity was more

significantly influenced by branching location, with core and peripheral multiplicity imparting opposing effects on self-assembly behavior. Amphiphiles with either high branching multiplicity at both the core and periphery or low branching multiplicity at both sites exhibit poor aggregate encapsulation capacity, whereas balancing branching between locations was seen to improve reverse micelle dye loading.

Finally, in vitro transdermal diffusion studies indicate that


33


Page 34 of 51

branching location influences carrier dermal permeation efficacy, beyond the topological effects on aggregate size or encapsulation capacity. In the case of tetra-laurylated amphiphiles C1(PEG-[G2]-LE4)1 and C4-(PEG-LE)4, the 1-arm species (with branching only at the periphery) was more successful at transporting dye into the skin than the 4-arm carrier analog (with branching only at the core), despite the later having a greater payload capacity. By physical characterization, these two equally-branched polymers exhibit similar self-assembly properties, such as size and CMC; nonetheless, carrier-mediated dye permeation of C1-(PEG-[G2]-LE4)1 was 6.5-fold greater in the VE than for C4-(PEG-LE)4. To the extent that more general structural conclusions may be drawn from this limited set of systematically branched amphiphiles, a transdermal drug delivery carrier may benefit from increased branching specifically at its periphery.

ASSOCIATED CONTENT Supporting Information. Spectroscopy and transdermal diffusion control experiments and related data, additional amphiphile UV absorbance and fluorescence spectra and plots, transdermal experiment data and results for secondary carrier concentrations. ACKNOWLEDGEMENTS We thank the National Science Foundation for their financial support of this research (CHE1412439, IAA-1430280) and graduate fellowship funding (KAK, SLK, Bioinnovation IGERT, DGE-1144646). We also acknowledge the laboratory of Dr. Russell Schmehl, Rebecca Adams, Stephen Guertin and Aditya Kulkarni (Tulane Chemistry) and extend gratitude for use of their spectroscopy instruments and for guidance in data acquisition.


34

Page 35 of 51 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

Biomacromolecules

REFERENCES (1) Cevc, G.; Vierl, U. Nanotechnology and the Transdermal Route: A State of the Art Review and Critical Appraisal. J. Controlled Release 2010, 141, 277-299. (2) Prausnitz, M. R.; Mitragotri, S.; Langer, R. Current Status and Future Potential of Transdermal Drug Delivery. Nat. Rev. Drug Discovery 2004, 3, 115-124. (3) Thummel, K. E.; O'Shea, D.; Paine, M. F.; Shen, D. D.; Kunze, K. L.; Perkins, J. D.; Wilkinson, G. R. Oral First‐Pass Elimination of Midazolam Involves Both Gastrointestinal and Hepatic CYP3A‐Mediated Metabolism. Clin. Pharmacol. Ther. 1996, 59, 491-502. (4) Pegoraro, C.; MacNeil, S.; Battaglia, G. Transdermal Drug Delivery: From Micro to Nano. Nanoscale 2012, 4, 1881-1894. (5) Scheuplein, R. J.; Blank, I. H. Permeability of the Skin. Physiol. Rev. 1971, 51, 702-747. (6) Downing, D. T. Lipid and Protein Structures in the Permeability Barrier of Mammalian Epidermis. J. Lipid Res. 1992, 33, 301-313. (7) Bos, J. D.; Meinardi, M. H. M. The 500 Dalton Rule for the Skin Penetration of Chemical Compounds and Drugs. Exp. Dermatol. 2000, 9, 165-169. (8) Roy, S. D.; Flynn, G. L. Transdermal Delivery of Narcotic Analgesics: pH, Anatomical, and Subject Influences on Cutaneous Permeability of Fentanyl and Sufentanil. Pharm. Res. 1990, 7, 842-847. (9) Yuan, X.; Capomacchia, A. C. Influence of Physicochemical Properties on the In Vitro Skin Permeation of the Enantiomers, Racemate, and Eutectics of Ibuprofen for Enhanced Transdermal Drug Delivery. J. Pharm. Sci. 2013, 102, 1957-1969. (10) Potts, R. O.; Guy, R. H. Predicting Skin Permeability. Pharm. Res. 1992, 9, 663-669. (11) Chandrashekar, N.; Rani, R. S. Physicochemical and Pharmacokinetic Parameters in Drug Selection and Loading for Transdermal Drug Delivery. Indian J. Pharm. Sci. 2008, 70, 94. (12) Prausnitz, M. R.; Langer, R. Transdermal Drug Delivery. Nat. Biotechnol. 2008, 26, 12611268. (13) Prausnitz, M. R. Microneedles for Transdermal Drug Delivery. Adv. Drug Delivery Rev. 2004, 56, 581-587. (14) Donnelly, R. F.; Singh, T. R. R. Novel Delivery Systems for Transdermal and Intradermal Drug Delivery; John Wiley & Sons: New York, 2015.


35


Page 36 of 51

(15) Prow, T. W.; Grice, J. E.; Lin, L. L.; Faye, R.; Butler, M.; Becker, W.; Wurm, E. M.; Yoong, C.; Robertson, T. A.; Soyer, H. P. Nanoparticles and Microparticles for Skin Drug Delivery. Adv. Drug Delivery Rev. 2011, 63, 470-491. (16) Hansen, S.; Lehr, C. M. Nanoparticles for Transcutaneous Vaccination. Microb. Biotechnol. 2012, 5, 156-167. (17) Wang, Y.-X.; Robertson, J. L.; Spillman, J. W. B.; Claus, R. O. Effects of the Chemical Structure and the Surface Properties of Polymeric Biomaterials on their Biocompatibility. Pharm. Res. 2004, 21, 1362-1373. (18) Barry, B. W. Novel Mechanisms and Devices to Enable Successful Transdermal Drug Delivery. Eur. J. Pharm. Sci. 2001, 14, 101-114. (19) Williams, A. Transdermal and Topical Drug Delivery: From Theory to Clinical Practice; Pharmaceutical Press: London, 2003. (20) Baroli, B. Penetration of Nanoparticles and Nanomaterials in the Skin: Fiction or Reality? J. Pharm. Sci. 2010, 99, 21-50. (21) Christophers, E. Cellular Architecture of the Stratum Corneum. J. Invest. Dermatol. 1971, 56, 165-169. (22) Johnson, M. E.; Blankschtein, D.; Langer, R. Evaluation of Solute Permeation Through the Stratum Corneum: Lateral Bilayer Diffusion as the Primary Transport Mechanism. J. Pharm. Sci. 1997, 86, 1162-1172. (23) Honeywell-Nguyen, P. L.; Gooris, G. S.; Bouwstra, J. A. Quantitative Assessment of the Transport of Elastic and Rigid Vesicle Components and a Model Drug from These Vesicle Formulations into Human Skin In Vivo. J. Invest. Dermatol. 2004, 123, 902-910. (24) Hillery, A. M.; Park, K. Drug Delivery: Fundamentals and Applications, 2nd ed.; CRC Press: Boca Raton, FL, 2016. (25) Kataoka, K.; Harada, A.; Nagasaki, Y. Block Copolymer Micelles for Drug Delivery: Design, Characterization and Biological Significance. Adv. Drug Delivery Rev. 2001, 47, 113-131. (26) Li, J.; Zhai, Y.; Zhang, B.; Deng, L.; Xu, Y.; Dong, A. Methoxy Poly(ethylene glycol)‐ block‐Poly(D,L‐lactic acid) Copolymer Nanoparticles as Carriers for Transdermal Drug Delivery. Polym. Int. 2008, 57, 268-274. (27) Yang, Y.; Sunoqrot, S.; Stowell, C.; Ji, J.; Lee, C.-W.; Kim, J. W.; Khan, S. A.; Hong, S. Effect of Size, Surface Charge, and Hydrophobicity of Poly(amidoamine) Dendrimers on their Skin Penetration. Biomacromolecules 2012, 13, 2154-2162.


36

Page 37 of 51 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

Biomacromolecules

(28) Poree, D. E.; Giles, M. D.; Lawson, L. B.; He, J.; Grayson, S. M. Synthesis of Amphiphilic Star Block Copolymers and their Evaluation as Transdermal Carriers. Biomacromolecules 2011, 12, 898-906. (29) Liu, H.; Jiang, A.; Guo, J.; Uhrich, K. E. Unimolecular Micelles: Synthesis and Characterization of Amphiphilic Polymer Systems. J. Polym. Sci., Part A: Polym. Chem. 1999, 37, 703-711. (30) Djordjevic, J.; Michniak, B.; Uhrich, K. E. Amphiphilic Star-Like Macromolecules as Novel Carriers for Topical Delivery of Nonsteroidal Anti-Inflammatory Drugs. AAPS J. 2003, 5, 1-12. (31) Kosakowska, K. A.; Casey, B. K.; Albert, J. N. L.; Wang, Y.; Ashbaugh, H. S.; Grayson, S. M. Synthesis and Self-Assembly of Amphiphilic Star/Linear-Dendritic Polymers: Effect of Core-vs-Peripheral Branching on Reverse Micelle Aggregation. Biomacromolecules 2018, in press. (32) Schneider, C. A.; Rasband, W. S.; Eliceiri, K. W. NIH Image to ImageJ: 25 Years of Image Analysis. Nat. Methods 2012, 9, 671. (33) Cliceti, P.; Veronese, F. M. Pharmacokinetic and Biodistribution Properties of Poly(ethylene glycol)-Protien Conjugates. Adv. Drug Delivery Rev. 2003, 55, 1261-1277. (34) Feliu, N.; Walter, M. V.; Montañez, M. I.; Kunzmann, A.; Hult, A.; Nyström, A.; Malkoch, M.; Fadeel, B. Stability and Biocompatibility of a Library of Polyester Dendrimers in Comparison to Polyamidoamine Dendrimers. Biomaterials 2012, 33, 1970-1981. (35) Jones, M. C.; Gao, H.; Leroux, J. C. Reverse Polymeric Micelles for Pharmaceutical Applications. J. Controlled Release 2008, 132, 208-215. (36) Torchilin, V. P. Structure and Design of Polymeric Surfactant-Based Drug Delivery Systems. J. Controlled Release 2001, 73, 137-172. (37) Riess, G. Micellization of Block Copolymers. Prog. Polym. Sci. 2003, 28, 1107-1170. (38) Alexandridis, P.; Lindman, B. Amphiphilic Block Copolymers: Self-Assembly and Applications; Elsevier: Amsterdam, 2000. (39) Vutukuri, D. R.; Basu, S.; Thayumanavan, S. Dendrimers with Both Polar and Apolar Nanocontainer Characteristics. J. Am. Chem. Soc. 2004, 126, 15636-15637. (40) Kalyanasundaram, K.; Thomas, J. Environmental Effects on Vibronic Band Intensities in Pyrene Monomer Fluorescence and their Application in Studies of Micellar Systems. J. Am. Chem. Soc. 1977, 99, 2039-2044. (41) Kumar, K. S.; Selvaraju, C.; Malar, E. J. P.; Natarajan, P. Existence of a New Emitting Singlet State of Proflavine: Femtosecond Dynamics of the Excited State Processes and Quantum Chemical Studies in Different Solvents. J. Phys. Chem. A 2011, 116, 37-45.


37


Page 38 of 51

(42) Hawker, C. J.; Wooley, K. L.; Fréchet, J. M. J. Solvatochromism as a Probe of the Microenvironment in Dendritic Polyethers: Transition from an Extended to a Globular Structure. J. Am. Chem. Soc. 1993, 115, 4375-4376. (43) Chambers, R. W.; Kajiwara, T.; Kearns, D. R. Effect of Dimer Formation on the Electronic Absorption and Emission Spectra of Ionic Dyes. Rhodamines and Other Common Dyes. J. Phys. Chem. 1974, 78, 380-387. (44) Wilhelm, M.; Zhao, C. L.; Wang, Y.; Xu, R.; Winnik, M. A.; Mura, J. L.; Riess, G.; Croucher, M. D. Poly(styrene-ethylene oxide) Block Copolymer Micelle Formation in Water: A Fluorescence Probe Study. Macromolecules 1991, 24, 1033-1040. (45) Astafieva, I.; Zhong, X. F.; Eisenberg, A. Critical Micellization Phenomena in Block Polyelectrolyte Solutions. Macromolecules 1993, 26, 7339-7352. (46) Creutz, S.; Van Stam, J.; De Schryver, F. C.; Jérôme, R. Dynamics of Poly((dimethylamino)alkyl methacrylate-block-sodium methacrylate) Micelles. Influence of Hydrophobicity and Molecular Architecture on the Exchange Rate of Copolymer Molecules. Macromolecules 1998, 31, 681-689. (47) Attwood, D.; Booth, C.; Yeates, S. G.; Chaibundit, C.; Ricardo, N. M. Block Copolymers for Drug Solubilisation: Relative Hydrophobicities of Polyether and Polyester Micelle-CoreForming Blocks. Int. J. Pharm. 2007, 345, 35-41. (48) Nagarajan, R.; Ganesh, K. Block Copolymer Self‐Assembly in Selective Solvents: Spherical Micelles with Segregated Cores. J. Chem. Phys. 1989, 90, 5843-5856. (49) Atanase, L. I.; Riess, G. Self-Assembly of Block and Graft Copolymers in Organic Solvents: An Overview of Recent Advances. Polymers 2018, 10, 62. (50) Khougaz, K.; Zhong, X. F.; Eisenberg, A. Aggregation and Critical Micelle Concentrations of Polystyrene-b-Poly(sodium acrylate) and Polystyrene-b-Poly(acrylic acid) Micelles in Organic Media. Macromolecules 1996, 29, 3937-3949. (51) Owen, S. C.; Chan, D. P.; Shoichet, M. S. Polymeric Micelle Stability. Nano Today 2012, 7, 53-65. (52) Trappmann, B.; Ludwig, K.; Radowski, M. R.; Shukla, A.; Mohr, A.; Rehage, H.; Böttcher, C.; Haag, R. A New Family of Nonionic Dendritic Amphiphiles Displaying Unexpected Packing Parameters in Micellar Assemblies. J. Am. Chem. Soc. 2010, 132, 11119-11124. (53) Qiao, H.; Li, J.; Wang, Y.; Ping, Q.; Wang, G.; Gu, X. Synthesis and Characterization of Multi-Functional Linear-Dendritic Block Copolymer for Intracellular Delivery of Antitumor Drugs. Int. J. Pharm. 2013, 452, 363-373. (54) d'Arcy, R.; Gennari, A.; Donno, R.; Tirelli, N. Linear, Star, and Comb Oxidation‐ Responsive Polymers: Effect of Branching Degree and Topology on Aggregation and Responsiveness. Macromol. Rapid Commun. 2016, 37, 1918-1925.


38

Page 39 of 51 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

Biomacromolecules

(55) Huh, J.; Kim, K. H.; Ahn, C.-H.; Jo, W. H. Micellization Behavior of Star-Block Copolymers in a Selective Solvent: A Brownian Dynamics Simulation Approach. J. Chem. Phys. 2004, 121, 4998-5004. (56) Haugen, G.; Melhuish, W. Association and Self-Quenching of Proflavine in Water. Trans. Faraday Soc. 1964, 60, 386-394. (57) Hurter, P. N.; Hatton, T. A. Solubilization of Polycyclic Aromatic Hydrocarbons by Poly(ethylene oxide-propylene oxide) Block Copolymer Micelles: Effects of Polymer Structure. Langmuir 1992, 8, 1291-1299. (58) Zou, J.; Zhao, Y.; Shi, W. Encapsulation Mechanism of Molecular Nanocarriers Based on Unimolecular Micelle Forming Dendritic Core−Shell Structural Polymers. J. Phys. Chem. B 2006, 110, 2638-2642. (59) Kumar, V.; Prud'Homme, R. K. Thermodynamic Limits on Drug Loading in Nanoparticle Cores. J. Pharm. Sci. 2008, 97, 4904-4914. (60) Gao, H.; Jones, M. C.; Tewari, P.; Ranger, M.; Leroux, J. C. Star-Shaped Alkylated Poly(glycerol methacrylate) Reverse Micelles: Synthesis and Evaluation of Their Solubilizing Properties in Dichloromethane. J. Polym. Sci., Part A: Polym. Chem. 2007, 45, 2425-2435. (61) Zou, J.; Zhao, Y.; Shi, W. Encapsulation Mechanism of Molecular Nanocarriers Based on Unimolecular Micelle Forming Dendritic Core− Shell Structural Polymers. J. Phys. Chem. B 2006, 110, 2638-2642. (62) Swindle, M.; Makin, A.; Herron, A.; Clubb Jr, F.; Frazier, K. Swine as Models in Biomedical Research and Toxicology Testing. Vet. Pathol. 2012, 49, 344-356. (63) Alvarez-Román, R.; Naik, A.; Kalia, Y.; Fessi, H.; Guy, R. H. Visualization of Skin Penetration Using Confocal Laser Scanning Microscopy. Eur. J. Pharm. Biopharm. 2004, 58, 301-316. (64) Labouta, H. I.; El-Khordagui, L. K.; Kraus, T.; Schneider, M. Mechanism and Determinants of Nanoparticle Penetration Through Human Skin. Nanoscale 2011, 3, 4989-4999. (65) Tormoehlen, L.; Tekulve, K.; Nanagas, K. Hydrocarbon Toxicity: A Review. Clin. Toxicol. 2014, 52, 479-489. (66) Das Kurmi, B.; Tekchandani, P.; Paliwal, R.; Rai Paliwal, S. Transdermal Drug Delivery: Opportunities and Challenges for Controlled Delivery of Therapeutic Agents Using Nanocarriers. Curr. Drug Metab. 2017, 18, 481-495. (67) Mignani, S.; El Kazzouli, S.; Bousmina, M.; Majoral, J.-P. Expand Classical Drug Administration Ways by Emerging Routes Using Dendrimer Drug Delivery Systems: A Concise Overview. Adv. Drug Delivery Rev. 2013, 65, 1316-1330.


39


Page 40 of 51

(68) Hood, R. R.; Kendall, E. L.; Junqueira, M.; Vreeland, W. N.; Quezado, Z.; Finkel, J. C.; DeVoe, D. L. Microfluidic-Enabled Liposomes Elucidate Size-Dependent Transdermal Transport. PLoS One 2014, 9, e92978. (69) Adib, Z. M.; Ghanbarzadeh, S.; Kouhsoltani, M.; Khosroshahi, A. Y.; Hamishehkar, H. The Effect of Particle Size on the Deposition of Solid Lipid Nanoparticles in Different Skin Layers: A Histological Study. Avd. Pharm. Bull. 2016, 6, 31. (70) Venuganti, V. V.; Sahdev, P.; Hildreth, M.; Guan, X.; Perumal, O. Structure-Skin Permeability Relationship of Dendrimers. Pharm. Res. 2011, 28, 2246.


40

Page 41 of 51 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

Biomacromolecules

FOR TABLE OF CONTENTS USE ONLY “Evaluation of amphiphilic star/linear-dendritic polymer reverse micelles for transdermal drug delivery: directing carrier properties by tailoring core-vs-peripheral branching” Karolina A. Kosakowska, Brittany K. Casey, Samantha L. Kurtz, Louise B. Lawson, Scott M. Grayson


41


Scheme 1. Chemical structures of the investigated branched amphiphiles, featuring control over the extent of branching at the core (vertical axis) and periphery (horizontal axis). 283x170mm (96 x 96 DPI)


Page 42 of 51

Page 43 of 51 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

Biomacromolecules

Figure 2. Branched amphiphile CMC (mM, log scale) values as obtained by fluorescence (solid line) and UV absorbance (dashed line) spectroscopy; (a) by core branching (x-axis); (b) by peripheral branching (xaxis); (c) 3-D bar graph of CMC (mM, via fluorescence) against both core (y-axis) and peripheral (x-axis) branching. 131x173mm (96 x 96 DPI)



Figure 3. Branched amphiphile reverse micelle CMC values (mM, top plot) and comparison of spectroscopy measured proflavine HS intensity per polymer concentration (I mM-1, values normalized relative to C4(PEG-LE)4 intensities, bottom plot) as observed by fluorescence (dark fill) and UV absorption (light fill), grouped by equivalent total branching number/amphiphile polarity ratio; (a) 2 laurate ester functionalities; (b) 4 laurate ester functionalities; (c) 8 laurate ester functionalities. 278x67mm (96 x 96 DPI)


Page 44 of 51

Page 45 of 51 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

Biomacromolecules

Figure 4. Summary of branched amphiphile library dye encapsulation results based on UV-vis absorption data, plotted with standard deviation as the mass ratio (wt./wt., mg g-1) of encapsulated proflavine HS mass per polymer mass; (a) by core branching (x-axis); (b) by peripheral branching (x-axis); and (c) as a bubble plot with the circle area depicting relative dye encapsulation as a function of both core (y-axis) and peripheral (x-axis) branching. 128x144mm (96 x 96 DPI)



Figure 5. Proflavine HS encapsulation capacity of branched amphiphile reverse micelles determined by UVvis measurement (n ≧ 3), plotted as a weight ratio of dye per polymer (wt./wt., mg g-1) and grouped by equivalent amphiphile polarity ratio/total number of peripheral groups; (a) 2 laurate ester functionalities; (b) 4 laurate ester functionalities; (c) 8 laurate ester functionalities. 275x43mm (96 x 96 DPI)


Page 46 of 51

Page 47 of 51 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

Biomacromolecules

Figure 6. Representative fluorescence microscopy images of porcine dermis cross-sections (SC up) following 90 min exposure to 5.0 mg mL-1 proflavine HS-loaded carrier solution of (a) C1-(PEG-LE)1; (b) C1-(PEG[G1]-LE2)1; (c) C1-(PEG-[G2]-LE4)1; (d) C2-(PEG-LE)2; (e) C2-(PEG-[G1]-LE2)2; (f) C2-(PEG-[G2]LE4)2; (g) C4-(PEG-LE)4; (h) C4-(PEG-[G1]-LE2)4; (i) C4-(PEG-[G2]-LE4)4; right-hand panel shows green channel (λex = 488 nm) capture only; left-hand panel shows overlay of green and blue (λex = 405 nm) channels (nuclei stained blue with DAPI). 271x274mm (96 x 96 DPI)



Figure 7. Background corrected fluorescence intensity measured in the SC (top), VE (middle) and UD (bottom) of carrier-treated porcine dermis, plotted by (a) core branching; (b) peripheral branching. Values were averaged from multiple tissue sections (n > 3) and corrected for autofluorescence. 128x114mm (96 x 96 DPI)


Page 48 of 51

Page 49 of 51 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

Biomacromolecules

Figure 8. Relative fluorescence intensity of carrier-treated tissue in the SC (top), VE (middle) and UD (bottom) adjusted per aggregate encapsulation capacity, plotted by (a) core branching; (b) peripheral branching. 128x106mm (96 x 96 DPI)



Figure 9. Effect of aggregate size on relative fluorescence intensity of carrier-treated tissue in the SC (top), VE (middle) and UD (bottom) adjusted per aggregate encapsulation capacity, plotted as a function of diameter (nm, x-axis) in series of (a) peripheral branching; (b) core branching; (c) all branched amphiphile carriers, distinguishing between peripheral (red, yellow, blue) and core (no pattern, sparse pattern, dense pattern) variability. 128x171mm (96 x 96 DPI)


Page 50 of 51

LinearâDendritic Polymer Reverse

Recommend Documents

LinearâDendritic Polymer Reverse