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Local and sustained activity of doxycycline delivered with layer-by-layer microcapsules Dong Luo, David James Gould, and Gleb B. Sukhorukov Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00070 • Publication Date (Web): 11 Mar 2016 Downloaded from http://pubs.acs.org on March 15, 2016
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Biomacromolecules
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Local and sustained activity of doxycycline
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delivered with layer-by-layer microcapsules
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Dong Luo1, David J Gould2*‡, Gleb B. Sukhorukov1*‡
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1
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4NS, UK
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2
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Keywords: antibiotic, doxycycline, layer-by-layer, microcapsules, drug
School of Engineering & Materials Science, Queen Mary University of London, London, E1
Bone & Joint Research Unit, Queen Mary University of London, London, UK
8
9
ABSTRACT
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Achieving localized delivery of small molecule drugs has the potential to increase efficacy and
11
reduce off target and side effects associated with systemic distribution. Herein, we explore the
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potential use of layer-by-layer (LbL) assembled microcapsules for the delivery of doxycycline.
13
Absorbance of doxycycline onto core dextran sulphate of pre-assembled microcapsules provides
14
an efficient method to load both synthetic and biodegradable microcapsules with the drug.
15
Application of an outer layer lipid coat enhances the sustained in vitro release of doxycycline
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from both microcapsule types. To monitor doxycycline delivery in a biological system C2C12
17
mouse myoblasts are engineered to express EGFP under the control of the optimized components
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of the tetracycline regulated gene expression system. Microcapsules are not toxic to these cells
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and upon delivery to the cells, EGFP is more efficiently induced in those cells that contain
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engulfed microcapsules and monitored EGFP expression clearly demonstrates that synthetic
4
microcapsules with a DPPC coat are the most efficient for sustain intracellular delivery.
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Doxycycline released from microcapsules also displayed sustained activity in an antimicrobial
6
growth inhibition assay compared with doxycycline solution. This study reveals the potential for
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LbL microcapsules in small molecule drug delivery and their feasible use for achieving
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prolonged doxycycline activity.
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Introduction
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Polyelectrolyte microcapsules generated on a dissolvable matrix core using the layer-by-layer
11
(LbL) assembly method were first described in 1998.1 Their construction utilizes electrostatic
12
interactions between oppositely charged polyelectrolytes to add layers to the core. Assembly is
13
performed under native conditions which permit the inclusion of biologically active molecules
14
within the structure, the positioning of these, influences activity in intact capsules as well as
15
stability and release kinetics.2, 3 The inclusion of metal nanoparticles as a charged species in
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microcapsule layers enables control over movement (magnetism)4 and can provide a remote
17
mechanism to release microcapsule contents.5-7 Microcapsules have been extensively studied in
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isolation and have been successfully employed for the delivery of macromolecules to cells, when
19
they are multifunctionalised they can be utilized in more intricate applications such as ATP
20
synthesis8 and biosensors.9 LbL microcapsules are usually assembled on a calcium carbonate
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core which yields microcapsules that are 3 to 4 µm in diameter. Their size has implications for
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their use in cellular systems, firstly, they will be engulfed by phagocytes which facilitates their
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use in antigen delivery,10 secondly, if they are delivered to extravascular compartments of the
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body they will not enter the blood and will be retained within the delivered tissue unless removed
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mobile cells. Their fate within cells depends upon their structure that those composed of
4
biodegradable polymers will be broken down and release their contents whilst those assembled
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with synthetic polymers can potentially remain intact. LbL microcapsules can be used to deliver
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macromolecules to cells but the incorporation and delivery of small molecule drugs is more
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problematic because they can diffuse out of the capsule structure. Strategies that have been
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employed to encapsulate small molecule drugs include the use of precipitated11 or crystallized
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material in the capsule core12 and adsorbing molecules into the microcapsule structure after
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assembly.13 In addition other attributes such as heat shrinkage, crosslinking and outer lipid
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layers14 can be applied to promote retention of molecules.
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Small molecule drugs are usually delivered orally and distribute systemically when local
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pharmacological effects are actually required. Systemic distribution can cause ‘off target’,
14
adverse and side effects. If local delivery can be achieved such drugs could have greater efficacy
15
and improved safety. Doxycycline is a widely used antibiotic in the treatment of infections in
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humans. At low concentrations below that required for antimicrobial effects doxycycline also
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exerts anti-inflammatory effects due to inhibition of matrix metalloproteinases15 so has potential
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use in treatment of inflammatory conditions.16 Doxycycline is typically administered orally as
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tablets or suspensions and also topically in cream. There have been no previous reports on the
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incorporation of doxycycline into LbL microcapsules but doxycycline has been incorporated into
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both nano and microstructures however sustained biological activity of the antibiotic has not
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been explored in these studies.17, 18
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In this work, doxycycline was encapsulated into LbL microcapsules by forming a complex
2
with dextran sulphate sodium salt in the microcapsule core or structure and sustained effect was
3
monitored by both an intracellular assay and antimicrobial assay. The microcapsule design was
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optimized by coating microcapsules with lipid. The lipid coating altered the microcapsule
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appearance;,the release kinetics of doxycycline, and biological activity of doxycycline.
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Experimental section
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Materials
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Poly(allylamine hydrochloride) (PAH, 56 kDa), Poly(sodium 4-styrenesulfonate) (PSS, 70
9
kDa), Dextran sulphate sodium salt (DS, 100 kDa), Poly-L-arginine hydrochloride (Parg, 15–70
10
kDa), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),L-α-Phosphatidylcholine (lecithin,
11
from egg yolk), Doxycycline Hydrochloride, Rhodamine B isothiocyanate,
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Tetrazolium Bromide (MTT), Cell Lysis Reagent (CelLytic™ M ), Protease Inhibitor Cocktail,
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Ethylenediaminetetraacetic Acid disodium salt (EDTA), Calcium Chloride, Sodium Carbonate
14
and all other salts were purchased from Sigma-Aldrich UK.1, 2-dipalmitoyl-sn-glycero-3-
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phosphoethanolamine-N-(lissaminerhodamine B sulfonyl) (ammonium salt) was purchased from
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Avanti Polar-lipids. Agar (Fisher BioReagents) and LB Broth, Miller (Fisher BioReagents) were
17
used for antimicrobial studies. Other biological reagents, including DMEM, PBS, foetal calf
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serum, penicillin and trypsin were purchased from Lonza Biologics Inc (Newington, NH, United
19
States).
20
Microcapsule synthesis and doxycycline encapsulation
Thiazolyl Blue
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The layer-by-layer self-assembly technique was used for microcapsule construction on a
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CaCO3 sacrificial template for oppositely charged polyelectrolytes. For subsequent loading of
23
doxycycline into capsules, DS was co-precipitated within CaCO3 according to the method for
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encapsulation of large molecules3. DS (20 mg/ml) was added to 0.33 M CaCl2 solution and was
2
then immediately mixed with 0.33 M Na2CO3 by vigorous stirring for 20 seconds and then
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incubated for a further 10 seconds. The hybrid CaCO3 particles were washed three times with
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deionized H2O, and were immediately used for microcapsule assembly. PAH and PSS were used
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for synthetic shells, and Parg and DS as biodegradable ones. Due to the DS trapped in the
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templates, the cores were initially negatively charged so PAH or Parg was assembled as the first
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layer. All the polymer solutions were prepared at 2 mg/ml with NaCl (0.2 M), and after
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absorbing each layer, microcapsules were triple washed with deionized H2O. For fluorescent
9
visualization, the second positive layer was replaced with TRITC-PAH or TRITC-Parg. Six
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layers were deposited for all the capsules before dissolving the templates with 0.2 M EDTA
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solution.
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DS trapped in the hollow cavity of microcapsules was used as attraction for doxycycline
13
molecules. This method has been used for encapsulating soluble low molecular weight drugs,19
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which are loaded via electrostatic interaction or as a shell component. Herein, doxycycline
15
molecules penetrate the shells and react with DS and form a complex within the microcapsule.
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Microcapsules were counted with a haemocytometer and the concentration adjusted to 6×107
17
capsules per ml before mixing with 1 ml of doxycycline solution (10 mg/ml) and shaking for 8
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hours. At the end of this incubation maximum encapsulation was achieved by placing the
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mixture at 60℃ for 10 min to shrink the capsules according to a previously reported procedure.20
20
Then the capsules were washed three times with deionized water and supernatants were collected
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to determine amount of loaded doxycycline. In order to determine the amount of encapsulated
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doxycycline per gram of microcapsules, equal quantity of microcapsules (both empty capsules
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and doxycycline loaded ones) were freeze dried (ScanVac Cool Safe Freeze Drying, Denmark) at
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-107 ℃, 0.009 mBar for 2 days. Then each sample was weighed (CP2-P microbalance, Sartorius
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AG Gottingen, Germany) and encapsulated doxycycline weight was calculated.
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Lipid layer assembly
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Phospholipids, DPPC or lecithin, were coated as outmost layers on microcapsules to further
5
reduce the permeability and thereby reduce the doxycycline leakage. DPPC (for synthetic
6
capsules) and lecithin (for biodegradable capsules) were dissolved in chloroform at 0.5 mg/ml
7
and were then evaporated using a rota vap at 42℃, after which a thin layer of phospholipids was
8
formed.21, 22 Liposomes were then constructed by adding water (final lipid concentration of
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1mg/ml) to the lipid film and sonicating for 1 hour at 60℃. The formed lipid vesicles were added
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to the doxycycline loaded capsules and shaken for 20 min. Then the mixture was washed 3 times
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with deionized water to remove excessive lipid, and supernatants were also collected for
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doxycycline concentration measurement. To visualize the lipid coating, Rhodamine B labelled
13
lipid (0.2 mg/ml) was mixed with the DPPC or lecithin. Permeability of microcapsules was
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tested with FITC under the confocal microscope.
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Release profile of doxycycline from microcapsules
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Release of doxycycline from microcapsules was determined by a fluorescence spectrum assay.
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The doxycycline concentration in solution was calculated according to the emission fluorescence
18
intensity at 536 nm. Typically, a series of standard doxycycline solutions (40, 30, 20, 15, 10, 5,
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4, 3, 2, 1, 0.5, 0.25, 0.1, 0.05 ug/ml) were prepared and emission intensity at 536 nm was
20
measured (excited at 350nm). The calibration curve was used to determine amount of
21
doxycycline in supernatants collected during the assembly process and therefore enable
22
calculation of the amount of doxycycline retained in each capsule (Supplementary Figure S1).
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In the release experiments, 12×107of synthetic or biodegradable capsules were prepared in a 1
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ml suspension in either deionized water, PBS or NaCl (0.15M). The suspensions were shaken on
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a vortex for specified intervals, after which it was centrifuged and the supernatant was collected
4
for evaluation of the doxycycline released. The removed supernatant was replaced with 1 ml of
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fresh solution and the incubation continued. To demonstrate 100% release, doxycycline loaded
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microcapsules were mixed with 2 M NaOH.23 At this pH, microcapsules disassemble and the
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doxycycline concentration in the solution was measured.
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Engineering C2C12 myoblasts as doxycycline reporter cells
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The lentiviral vectors used to engineer mouse C2C12 myoblast cells (ATCC® CRL-1772™)
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were pLOX-TwGFP which encodes EGFP from the Ptet and pUCL-MIK which encodes the
11
rtTA-2SM2 transactivator24 and the tetRKRAB repressor25 from a bicistrontic cassette26 under
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the control of the constitutive SFFV promoter.27 Lentivirus was prepared as previously
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described28 then C2C12 cells were multiply infected with pUCL-MIK before multiple infection
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with pLOX-TwGFP. The resulting cell population was then stimulated with doxycycline
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hydrochloride (1 µg/ml) and after 24 hours the cells with the highest expression of EGFP were
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sorted using a BD FACS Aria II. These cells were plated on a 96 well plate at 1 cell per well and
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clones expanded. The selected clone was not fluorescent in the unstimulated state but
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ubiquitously expressed high levels of EGFP upon doxycycline stimulation.
19
MTT assay
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The myoblast cells were cultured in the Dulbecco's minimum essential media, containing 10%
21
fetal bovine serum (FBS) and 1% penicillin–streptomyosin, at 37 °C and 5% CO2. The standard
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MTT assay was used to assess the cell viability after treatment with doxycycline solution, empty
23
capsules and doxycycline-loaded capsules for 48 hours. Cells were seeded in 96-well plates with
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1×104 cells per well and capsules were added at 10:1 ratio. After 48 hours, medium was
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removed, 20 µl of MTT solution (in PBS) was added to each well and cells were cultured for 3.5
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hours. Then 150 µl MTT solvent (4 mM HCl, 0.1% Nondet P-40 in isopropanol) was added to
4
each well. The plate was read in a BMG Fluostar Galaxy plate reader (filter 570 nm).
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Cell uptake of doxycycline capsules and induced EGFP expression
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Cellular uptake of capsules and induced EGFP expression was visualized using a Leica
7
confocal microscope with 40× oil emersion objective. Cells were seeded on 8-well glass chamber
8
slides (well size 10.7 mm×9.4 mm, ibidi USA Inc) at 1×104 cells per well and 10 times the
9
number of doxycycline loaded capsules were added immediately. For the control, the equivalent
10
amount of free doxycycline was mixed with the medium. After 2 days, free capsules were
11
removed by washing with PBS, and fluorescence and phase contrast microscopy images were
12
taken.
13
To observe the induced EGFP expression, cells co-cultured with doxycycline-loaded capsules
14
for one day were trypsinized and centrifuged to remove any free or cell surface attached
15
capsules, and cells were re-seeded in the chamber. Eight hours later, images in green, red and
16
transmission channels were taken at the same time from one side of the chamber to the other to
17
form a large panoramic image. Cells were counted in the large image, and classified into four
18
groups, namely green and red (cells with both EGFP and capsules), green (cells with EGFP
19
only), red (cells with capsules only) and none (cell without EGFP or capsules).
20
In order to evaluate the EGFP expression kinetics, cells with both free doxycycline and
21
doxycycline-loaded capsules (10:1) were cultured in the chamber for 34 days, and confocal
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images were taken every two days. To reduce the effect of residual doxycycline, after 2 days
23
incubation with microcapsules medium was changed every day. These studies on EGFP
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expression enable comparison of expression kinetics for doxycycline in solution, doxycycline-
2
loaded synthetic capsules and biodegradable capsules.
3
EGFP fluorescence intensity assay
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Quantitative analysis of EGFP expression kinetics was performed with a Fluostar Galaxy plate
5
reader. Engineered C2C12 cells (1×104 cells per well) in a 96-well plate were treated with free
6
doxycycline, doxycycline-loaded synthetic or biodegradable capsules (7 wells for each group).
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After specific intervals (2, 4, 6, 8, 10, 12 days), culture medium were removed, and cells were
8
washed with PBS and lysed for 30 min by addition of 20 µl lysis reagent (CelLytic™ M, with
9
0.05% Protease Inhibitor Cocktail added) to each well. Then 80 µl PBS was added, and EGFP
10
fluorescence intensity was measured in the reader with excitation and emission wavelengths
11
fixed at 480 nm and 520 nm accordingly. Control lysates were prepared using untreated cells and
12
the assay blank consisted of lysis reagent and PBS up to 100 µl volume. EGFP intensity was
13
calculated according to the following equation, EGFP fluorescence intensity = (intensity of test
14
well – intensity of control well) / intensity of control well ×100%.
15
Bacterial Inhibition assay
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The antibacterial activity of doxycycline loaded microcapsules was tested by growth inhibition
17
of Escherichia coli (E. coli, DH5a). Bacterial suspensions in LB Broth were cultured at
18
37°C/200rpm shaking until they were in the linear phase of growth indicated by an optical
19
density between 0.4-0.6 measured using a spectrophotometer (Cecil CE2021) at 400nm.
20
Bacterial suspension (0.4 ml) was spread on the surface of dried LB agar plates. The sensitivity
21
of the E. coli to inhibition with doxycycline was firstly demonstrated using filters (diameter 7
22
mm) treated with doxycycline concentrations from 5 mg/ml to 8 µg/ml and placed on an agar
23
plate pre-spread with E. coli. Filters remained on the plate which was incubated at 37°C
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overnight and then diameter of zones of inhibition were measured in mm. Sustained release of
2
doxycycline from microcapsules was examined in a transfer experiment in which filters were
3
initially loaded with 40 µl of doxycycline solution (0.1 or 1 mg/ml) or 40 µl of microcapsule
4
suspension equivalent 1 mg/ml of doxycycline. After incubation for 90 minutes on an agar plate
5
pre-spread with E. Coli, filters were transferred to a second pre-spread plate with addition of 10
6
µl sterile water to the filter, the process was repeated every 90 minutes. After 12 hours, all the
7
filters were disposed and all the plates were incubated overnight at 37°C. Images were taken and
8
the inhibitory zones against E. coli growth were measured using Nano Measurer(1.2).
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Statistical Methods
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Descriptive statistics and significant differences between groups were calculated using the
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Student's t tests for two-sample data of unequal variance (Microsoft® Excel 07 software);
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significance refers to p values less than 0.05 and 0.01.
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Results and discussion
14
Doxycycline-loaded microcapsule preparation
15
Small molecule drugs can be incorporated into microcapsules through electrostatic interactions
16
following a post loading approach.29, 30 Doxycycline is a small molecule drug (structure shown in
17
Figure 1a), and was encapsulated by precipitating with DS (Supplementary Figure S2).
18
Typically, two types of capsule were assembled, synthetic capsules consisting of PAH/PSS
19
layers and biodegradable ones composed of Parg/DS, both of which had DS in the interior for
20
doxycycline loading (Figure 2a and2d). Upon incubation, doxycycline penetrated the
21
microcapsule layers forming a less soluble complex with DS in the core, as well as being trapped
22
within the layers. The synthetic capsules became smaller in size and had a more protuberant
23
structure after doxycycline loading (Figure 2b), indicating that doxycycline-DS complex was
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formed in the core. Whilst, the biodegradable capsules did not undergo such notable
2
morphological change, but according to the increased capsule shell thickness (Figure 2e), we
3
could assume that more doxycycline was absorbed in the microcapsule walls. The principle of
4
the complex formation is based on the ion paring ability of dextran sulphate, as indicated by the
5
FTIR spectrum (Figure 1b), which was also used to promote minocycline loading efficiency in
6
PLGA nanoparticles.31 Similar phenomenon were observed when ciprofloxacin was deposited
7
into capsules, with a black shadow seen in the capsule interior under TEM and an increase in
8
average capsule height by 500 nm.32 According to measurement of supernatants from
9
encapsulation, doxycycline loaded in synthetic and biodegradable capsules was determined to be
10
5.4 pg and 4.8 pg per capsule, respectively. Doxycycline amount per gram of capsules was
11
determined to be around 97.4 mg according to the net weight increase compared to the empty
12
microcapsules. The results were confirmed by fluorescence imaging, synthetic capsules with
13
doxycycline had more blue fluorescence in the core, while blue fluorescence in biodegradable
14
capsules was within the rings of microcapsule shells (Supplementary Figure S3) in both
15
instances doxycycline is complexed with DS. a
b Doxy DS DS-Doxy
-
-SO3
-OH
-NH
3500
3000
-C=O
4000
2500
2000
1500
1000
500
16
Figure 1. Doxycycline and FTIR spectrum. Molecular structure of doxycycline (a), FTIR
17
spectrum of doxycycline (blue), DS sodium salt (black) and doxycycline-DS complex (red) (b).
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1
Since the microcapsule shells are permeable, extra layers are often assembled to enable longer
2
drug retention. Such layers are either more hydrophobic or have a denser meshwork. The
3
phospholipids DPPC and lecithin were used as an outer layer for synthetic and biodegradable
4
capsules respectively. As could be seen by changes in zeta potential (Supplementary Figure S4),
5
the outmost DPPC coating altered the negative charge of PSS to slightly positive, which
6
confirmed addition of the lipid layer on the surface. In addition, confocal images of capsules
7
show red circles when the lipid was labelled with Rhodamine B. In the permeability test, the
8
outer lipid layer also effectively hindered penetration of FITC through capsules. Whilst an
9
incomplete lipid layer on the microcapsule surface only rendered them partially permeable to
10
FITC (Supplementary Figure S5). These results provide evidence that microcapsule permeability
11
was reduced by the lipid coating. SEM images of lipid coated capsules (Figure 2c and f) shows
12
that DPPC, or lecithin coating smoothed the capsule surface (inset image), which was evidenced
13
as a more compact outer shell was formed. Similar morphological changes were previously
14
observed when biotinylated lipid was assembled on the surface of PAH/PSS capsules resulting in
15
an uncollapsed structure.33 According to measurement of supernatants, doxycycline loaded in
16
DPPC-coated synthetic and lecithin-coated biodegradable capsules was 5.3 pg and 5.4 pg per
17
capsule, respectively.
18
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Figure 2. Microcapsule images. SEM images of synthetic capsules (a-c) and biodegradable
2
capsules (d-f), before being loaded with doxycycline (a, d), after loading with doxycycline (b, e),
3
and coated with a lipid layer on the surface (c, f) (scale bar of 20 µm); the inset images are
4
individual capsules at high magnification (×40000) (scale bar of 3 µm).
5 6
Release kinetics of doxycycline in different mediums
7
Release kinetics of a drug from LbL assembled microcapsules is primarily dependent on
8
physio-chemical properties of the polymeric shells34 and the binding characteristics between the
9
drug molecules and capsules.30, 35 In our study, doxycycline was trapped by forming a complex
10
with DS, and the release of payload was affected by both factors. Due to the porous structure of
11
polymeric shells, doxycycline was able to diffuse through, but the capsules shell thickness,
12
polyelectrolyte molecular weight and ionic strength of the medium all affect the diffusion rate.34
13
Changes in the ionic strength of solution alter the strength of electrostatic interactions within the
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microcapsule shell structure and in the binding between doxycycline and DS. By shaking in
2
water, 0.15M NaCl or PBS, doxycycline release from all microcapsules was monitored, and the
3
rate of doxycycline release from all microcapsules was PBS>0.15M NaCl>water (Figure 3). In
4
low ionic strength solution, a tightly packed microcapsule structure is observed, but when the
5
polymeric structure is transferred to high ionic strength solution, the charge of polymers is
6
shielded and the rearranged microcapsule structure is more permeable to small molecules.36 With
7
regards to doxycycline and DS binding, in water, there would be a stronger binding between the
8
two molecules, due to charge interaction of negatively charged DS and amino groups of
9
doxycycline, but PBS and NaCl might accelerate the dissociation of the DS-doxycycline
10
complex via charge decoupling in salt solution which facilitates release of free doxycycline from
11
the capsule interior. In general the release of doxycycline from microcapsules was bi-phasic; a
12
primary burst release within the first 40 minutes and a slower secondary release after 40 minutes
13
(Table 1). The first phase is relevant to detaching of more loosely bound doxycycline and the
14
second phase is salt induced dissolution of the complex between DS and doxycycline. In the case
15
of pure water these two phases were less distinct. The sustained release measured in these
16
solutions contrasts to the complete dissociation of microcapsules with 100% doxycycline release
17
observed in 2M NaOH (data not shown).
18
The release kinetics were however slower when microcapsules were coated with a lipid layer
19
as shown in Figure 3c and 3d. The retention of doxycycline with the lipid coated capsules can be
20
attributed to lipophilic nature of doxycycline37 and to the hydrophobic barrier formed at the
21
outmost capsule surface by phospholipids which would be less permeable aqueous solutions. A
22
phospholipid outer layer seal was previously employed in the encapsulation of the small
23
molecule drug daunorubincin.22 The difference in the release kinetics for lipid coated synthetic
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1
and biodegradable capsules may be due to the fact that lecithin, a natural phospholipid, is a
2
heterogeneous molecule with a range of molecular weights which may prohibit the formation of
3
a more compact coating layer observed with synthetic DPPC.
4 a
b in H2O in 0.15 M NaCl in PBS
120
80
doxycycline released (%)
doxycycline released (%)
in H2O in 0.15 M NaCl in PBS
100
100
80
60
40
60
40
20
20
0
0 0
c
50
100
150
200
250
300
time (min)
0
d
in H2O in 0.15 M NaCl in PBS
100
50
100
150
200
250
300
time (min)
in H2O in 0.15 M NaCl in PBS
100
80
doxycycline released (%)
80
doxycycline released (%)
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
60
40
20
60
40
20
0
0 0
50
100
150
200
250
300
0
50
100
time (min)
150
200
250
300
time (min)
5
Figure 3. Kinetics of doxycycline release from microcapsules. Release profile of doxycycline
6
from synthetic capsules (a), biodegradable capsules (b), synthetic capsules with DPPC coating
7
(c) and biodegradable capsules with lecithin coating (d) in water, 0.15 M NaCl, and PBS. Values
8
are the mean of 3 groups and vertical bars represent the SD.
9 10 11 12
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Table 1 Rate of doxycycline release rates from microcapsules in different solutions. Phase 1
2
release is the kineic in the first 40 minutes whilst phase 2 release relates to the kinetic iduring the
3
subsequent 235 minutes. Doxycycline release rate
In H2O
(ug/min)
Phase1 Phase2
Phase1 Phase2
Phase1 Phase2
Syn capsules
0.229
0.067
0.700
0.019
1.595
0.032
DPPC coated syn capsules
0.263
0.016
0.428
0.029
1.045
0.043
Bio capsules
0.305
0.043
0.769
0.021
1.794
0.039
Lecithin coated bio capsules
0.313
0.019
0.599
0.029
1.476
0.059
In NaCl
In PBS
4 5 6
Cytotoxicity of microcapsules
7
Viability of cells treated with doxycycline solution, synthetic and biodegradable capsules (w/o
8
doxycycline encapsulated) is shown in Figure 4. Compared to the control group, cells treated
9
with doxycycline solution (2.5 µg/ml) had a viability of 89% after 48 hours incubation. With
10
both synthetic and biodegradable capsules, over 80% cell viability was observed at the same time
11
point. In addition, no significant difference was observed between empty and doxycycline loaded
12
capsules which was consistent with previous studies of capsule cytotoxicity in vitro and in vivo.4,
13
38, 39
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w/o doxy with doxy 100
80
viability, %
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
60
40
20
0
control
syn capsules
bio capsules
1
Figure 4. Microcapsules cytotoxicity. Cell viability of engineered C2C12 cells incubated with
2
doxycycline solution, synthetic and biodegradable capsules with and without (w/o) doxycycline.
3
Values are the mean of six values and vertical bars represent the SD.
4
Induced expression of EGFP in C2C12 cells
5
When reporter cells were treated with doxycycline solution or encapsulated doxycycline the
6
different cell delivery routes impacts on the kinetics of EGFP expression from the tetracycline
7
responsive promoter (Ptet). The promoter is activated by rtTA-2SM2 in the presence of
8
doxycycline and is actively switched off upon removal of doxycycline through binding of the
9
repressor tetRKRAB. This tet-on system has shown high gene regulation efficiency in our
10
previous study,40 and this was confirmed here. Confocal images were taken to assess whether
11
doxycycline loaded capsules were internalized by myoblast cells and subsequently induce EGFP
12
expression in C2C12 cells via the tet-on system. Synthetic capsules were incubated with cells for
13
2 days at a ratio of 10 capsules per cell, and the same amount of free doxycycline was used as
14
control. As illustrated in Figure 5, EGFP expression was successfully switched on by
15
doxycycline solution with homogenous fluorescence in every cell (Figure 5A). However, when
16
delivered as cargo by microcapsules, doxycycline was released after cell uptake of capsules, so
17
cells without internalized capsules had no fluorescence as indicated by red circles in Figure 5B.
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Biomacromolecules
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The results highlighted that doxycycline delivery from microcapsules can be targeted to specific
2
cells via capsule internalization which then function in situ.
3
To evaluate the efficiency of doxycycline delivered by capsules, cells were classified into four
4
categories (Green/red, green, red and none – see methods for details) and the number of cells in
5
each category was counted within the panoramic confocal image (Supplementary Figure S6), and
6
was presented as a percentage in Figure 6. The capsule uptake ratio by cells was about 70%, and
7
doxycycline capsules induced EGFP expression was as high as 61.8% (shown as green and red in
8
the histogram), but there were a small proportion of these cells with red microcapsules that were
9
devoid of EGFP expression (8.9%). When no capsules were engulfed 18% of cells were not
10
green as expected, but 11% were fluorescent. There are potential explanations for this
11
observation, firstly, capsules were not visible in these cells (hidden under the nucleus); secondly,
12
after cell division a daughter cell could contain EGFP but not a microcapsule from the parent
13
cell; alternatively doxycycline could leak from extracellular microcapsules located nearby or in
14
other cells. A similar phenomenon was observed when EGFP was delivered as cargo in capsules
15
to A549 cells, when cells with no engulfed capsules had EGFP in the cytosol.41 As for the non-
16
fluorescent cells that had internalized microcapsules, the absence of fluorescence may be due to
17
capsules being devoid of doxycycline or containing an amount of doxycycline below the
18
threshold to induce EGFP expression, though the baseline for doxycycline to switch on gene
19
expression is known to be very low (1 pg/ml).40 Previous studies have tried to chemically modify
20
doxycycline to achieve spatial and temporal gene expression regulation,42,
21
doxycycline was inactivated in a ‘cage’ so that transcription was only promoted after photo-
22
activation. But, delivery by versatile microcapsule carriers is a preferable approach as minimum
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in which
Page 19 of 37
1
modification to molecular structure is introduced and side products of chemical reactions are not
2
generated.
3 4
Figure 5. Images of doxycycline induced EGFP expression. Cells treated with a solution of
5
doxycycline (A) and doxycycline-loaded synthetic capsules (B) (capsules to cells ratio 10:1,
6
incubate time 48 hours, red circles indicate cells that have no internalized capsules or EGFP
7
expression) (scale bar of 50 µm).
70
60
50
percentage/ %
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
40
30
20
10
0 red/green
green
red
none
color of cells
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Page 20 of 37
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Figure 6. Correlation of microcapsule delivery and EGFP expression in C2C12 cells. After
2
incubation with doxycycline-loaded synthetic microcapsules that were labelled red due to use of
3
TRITC labelled PAH cells exposed to doxycycline express EGFP. The distribution of cells in the
4
four categories (Red/green: cells with microcapsules and expressing EGFP; Green: cells
5
expressing EGFP but have no capsules; Red: cells with microcapsules have no EGFP expression;
6
None: neither red or green) was determined by microscopic examination with results expressed
7
as a percentage of all cells (total number 459).
8
Kinetics of EGFP expression
9
To demonstrate the sustained effect of doxycycline delivered by microcapsules, the kinetics of
10
EGFP expression was monitored by fluorescence intensity change in long-term experiments.
11
Four types of capsules, synthetic (PAH/PSS) and biodegradable (DS/Parg) were compared with
12
their lipid coated counterparts. EGFP fluorescence intensity at specific time intervals was
13
measured for each type of capsule. Cells treated with free doxycycline had the maximum
14
fluorescence intensity at day 2 and declined to baseline values after 10 days (Figure 7a). For all
15
the capsule-treated cells, the fluorescence intensity increased and peaked on the fourth day, then
16
fluorescence decreased gradually, and there were significant differences compared with the free
17
doxycycline treated cells after the sixth day (p< 0.05). Doxycycline molecules are lipophilic
18
enabling them to rapidly enter cells and induce gene expression, while microcapsules enter cells
19
following membrane adherence, phagocytosis and macropinocytosis mechanisms, and are finally
20
targeted to phagolysosomes38, 44 doxycycline released from microcapsules in these compartments
21
can still penetrate lipid membranes but clearly the kinetics of nuclear delivery are slower than the
22
free doxycycline. For the synthetic capsules, DPPC coating resulted in elevated and prolonged
23
EGFP expression compared with the uncoated microcapsules (p
