RESEARCH ARTICLE Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Potential Avoidance of Adverse Analgesic Effects Using a

Biologically Smart Hydrogel Capable of Controlled
Bupivacaine Release
FRANCESCA TARABALLI,1,2 SILVIA MINARDI,1,3 BRUNA CORRADETTI,1,4 IMAN K. YAZDI,1,5 MARTA A. BALLIANO,1
JEFFREY L. VAN EPS,1,6 MASSIMO ALLEGRI,2,7 ENNIO TASCIOTTI1
1

Department of Nanomedicine, Houston Methodist Research Institute, Houston, Texas 77030

Pain Therapy Service, University of Pavia-Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
3
Bioceramics and Bio-Hybrid Materials, National Research Council of Italy ISTEC, Faenza, Ravenna 48018, Italy
4
Department of Life and Environmental Sciences, Universit`a Politecnica delle Marche, Ancona 60131, Italy
5
Department of Biomedical Engineering, University of Houston, Houston, Texas
6
Department of Surgery, Houston Methodist Hospital, Houston, Texas 77030
7
Department of Clinic Surgical Pediatric and Diagnostic Sciences, University of Pavia, Pavia, Italy
2

Received 18 June 2014; revised 8 August 2014; accepted 29 August 2014

Published online 29 September 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24190
ABSTRACT: Acute pain remains a tremendous clinical and economic burden, as its prevalence and common narcotic-based treatments
are associated with poorer outcomes and higher costs. Multimodal analgesia portends great therapeutic promise, but rarely allows opioid
sparing, and new alternatives are necessary. Microparticles (MPs) composed of biodegradable polymers [e.g., poly(lactic-co-glycolic
acid) or PLGA] have been applied for controlled drug release and acute pain treatment research. However, foreign particles presence
within inflamed tissue may affect the drug release or targeting, and/or cause a secondary inflammatory reaction. We examined how small
alterations in the particulate nature of MPs affect both their uptake into and subsequent activation of macrophages. MPs composed of PLGA
and chitosan (PLGAChi) loaded with bupivacaine (BP) were engineered at different sizes and their opsonization by J774 macrophages
was assessed. Uptake of PLGAChi by macrophages was found to be size dependent, but they were not cytotoxic or proinflammatory in
effect. Moreover, encapsulation of MPs in a thermoresponsive loading gel (pluronic F-127) effectively prevented opsonization. Finally, MPs
displayed sustained, tunable release of BP up to 7 days. These results demonstrate our ability to develop a drug delivery system capable
C 2014 Wiley
of controlled release of local anesthetics to treat acute/subacute pain while concurrently avoiding enhanced inflammation. 
Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:37243732, 2014
Keywords: pain; drug delivery; controlled release; inflammation; biomaterials; microparticles; injectables

INTRODUCTION
Acute postoperative pain is a complex, still unresolved phenomenon strictly related to postoperative morbidity and to a
chronic postsurgical pain syndrome.1,2 The impact of uncontrolled acute or subacute pain is far-reaching as well, with
numerous psychosocial and functional consequences that
significantly impact overall patient quality of life.37 Regional
analgesia and multimodal therapy, significantly diminishing
opioid requirements, and adverse effects incidence have transformed postoperative care, improving patients outcome and
enhancing their recovery.813 Continuous peripheral local anesthetic administration is a rational approach to control pain as
part of such multimodal strategies to reduce both peripheral
nociceptive input (and central sensitization) and peripheral
neuroinflammatory response that could be related to severe
postoperative pain and increased risk of persistent postoperative pain.1416 Unfortunately, this approach could be affected
Correspondence to: Ennio Tasciotti (Telephone: +8327552548; Fax:
+7134417438; E-mail: etasciotti@houstonmethodist.org)
This article contains supplementary material available from the authors upon
request or via the Internet at http://onlinelibrary.wiley.com/.
Journal of Pharmaceutical Sciences, Vol. 103, 37243732 (2014)


C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association

3724

by some adverse effects such as risk of catheter displacement

and lack of efficacy and infectious risk, patient activity reduction because of the use of pump to administer drugs, device
malfunction, need to refill, and systemic toxicity.17
The development of local anesthetic platforms that provide
prolonged local analgesia has classically been hindered by several pitfalls: inadequate duration of drug action, the appearance of systemic or organ-specific toxicity, and adverse local
tissue reaction or inflammation.17,18 Novel delivery platforms
capable of providing such prolonged, postoperative analgesia
without these limitations are valuable and necessary.
Carrier systems have emerged as an attractive alternative
with their ability to promote all of the desirable characteristics
for a local anesthetic, including: long duration of action, selectivity for sensory rather than motor nerve block, and reduction
of systemic toxicity.14,1820 A variety of controlled-release formulations have been developed, such as polymeric microparticles (MPs)2124 or liposomes.2528 Such systems have extended
the duration of nerve block to varying degrees ranging from
hours to weeks, and some of the more basic, US FDA-approved
formulations are now being adopted clinically. Only one such
formulation is currently approved for clinical use [liposomal
TM
bupivacaine (BP), EXPAREL ], and its opioid-sparing effect
is limited to 72 h.27 Examples of MPs already FDA-approved

Taraballi et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:37243732, 2014

RESEARCH ARTICLE Pharmaceutics, Drug Delivery and Pharmaceutical Technology

for use include poly-e-caprolactone and poly(lactic-co-glycolic

acid) (PLGA), but they can cause secondary inflammatory
reactionsthe nature of which appears strictly dependent on
the type of material and size of the particles.2932 Large particle size (20 :m) promotes chronic inflammatory infiltrate with
prominent foreign body giant cells, whereas smaller ones elicit
a phagocytic and lymphocytic response only. Thus, the process
of opsonization is one of the most important biological barriers to controlled drug delivery. The foreign body reaction and
chronic inflammatory state can affect the final drug efficacy
and release kinetics of different particle-loaded drugs.30,3335
In this work, we aimed to develop an environmentally sensible delivery system capable of prolonged local anesthetic (BP)
release, while avoiding foreign body reaction and cellular toxicity. We created PLGA MPs with a chitosan (Chi) coating because
it offers several advantages, including: intrinsic antibacterial
activity,36 human biocompatibility (minimizes local inflammation and foreign body reaction), biodegradability,37,38 enhanced
hemostasis,23,39 and ability to swell at acidic pH.37 In the acidic
extracellular matrix of inflamed tissue, Chi is fully protonated
and when it swells, releases the entrapped drug. Moreover, we
hypothesized that the incorporation of particles inside a thermoresponsive gel, pluronic F-127, will reduce the undesired local foreign body reaction, effectively cloaking the particles.40,41

MATERIALS AND METHODS

Bupivacaine hydrochloride, poly(vinyl alcohol), Rhodamine B,
Chi (LMW), Pluronic F-127, and all other reagents used were of
analytical grade, and were purchased from SigmaAldrich (St.
Louis, Missouri). PLGA-ester-terminated (lactideglycolide =
50:50) viscosity range 0.951.20 dl was purchased from LACTEL (Pelham, Alabama).
Preparation of PLGAChiBP Particles
Bupivacaine-loaded PLGAChi MPs were prepared by a
modified single emulsion method as mentioned in our previous studies.42 Briefly, PLGA (50:50) was dissolved in
dichloromethane (DCM) to form 10% (w/v) PLGADCM solution. Twenty milligrams of BP or 1 mg of Rhodamine B was
added while sonicating the mixture. The organic phase of the
mixture containing the drug was mixed with polyvinyl alcohol
2.5% (w/v) and (0.5%) Chi by vortex mixing and sonication. The
resulting suspension was stirred with a magnetic stir bar for
4 h and the DCM was eliminated by evaporation. BP was also
loaded on the outside Chi layer in order to synthesize PLGA
BPChiBP MPs. Briefly, 10 mg of BP was loaded under acidic
conditions (buffer acetate pH 5.5) in the Chi layer for 6 h at room
temperature; the suspension was washed three times with ultrapure water by centrifugation at 4424g for 10 min, then the
loaded MPs were freeze-dried and stored at 80 C for later use.
Characterization of PLGA MPs
Dynamic light scattering (DLS) was performed using a Zetasizer ZEN3600 (Malvern, Worcestershire, UK). For DLS, scattered light detection was measured at 90 to the incident beam
(a 25 mW laser at 660 nm wavelength). MPs size distribution
was measured using a Beckman Multisizer 3 Coulter Counter
(Beckman Coulter, Brea, CA). Fourier transformed infrared
(FT-IR) spectroscopy was performed by creating a pellet of 5%
sample and 95% KBr (SigmaAldrich) by volume and analyzDOI 10.1002/jps.24190

3725

ing absorbance of the pellet on a Nicolet 6700 FT-IR Spectrometer (ThermoFisher Scientific Inc, Walthman, MA). The
spectra were reported after background subtraction, baseline
correction, and binomial smoothing (11 points) using OMNIC
software (ThermoFisher Scientific Inc, Walthman, MA). The
morphology of the MPs was characterized by optical microscope
(Nikon Eclipse TS 100, Nikon Instrument Inc., Melville, NY),
fluorescent microscope (Nikon Eclipse TE 2000-E, Nikon Instrument Inc., Melville, NY), confocal laser microscope (Leica
MD 6000, Leica Microsystem, Inc., Buffalo Groove, IL), and
finally scanning electron microscope (SEM) (FEI Quanta 400
ESEM FEG, FEI, Hillsboro, OR) under a voltage of 5 KV.
Samples were sputtered with platinum (5 nm) by a Plasma
Sciences CrC-150 Sputtering System (Torr International, Inc.,
New Winsdor, NY) before SEM analysis.
Cytoxicity Analysis of PLGAChi Particles
The effect of empty PLGAChi MPs on the viability of cells was
determined by an Alamar Blue assay.43 Bone-marrow-derived
mesenchymal stem cells (mBM-MSCs) from mouse were isolated as previously reported for other species,44,45 and the absence of mycoplasma contamination was determined using the
MycoAlert mycoplasma detection kit (Lonza, Rockland, Maine).
The cells (105 cells/well) were cultured on 24-well plates with
500 :L of Dulbeccos modified Eagles medium/well. The mBMMSCs were treated with 50 :g/mL MPs and cellular viability was evaluated at days 1, 3, 5, and 7. Cellular metabolic
TM
assay (Invitactivity was quantified via the AlamarBlue
rogen, Carlsbad, CA). At each time point, culture medium
was removed and replaced with 500 :L of complete medium
TM
reagent and incubated for 2 h at
with 10% AlamarBlue

37 C, 5% CO2 . Absorbance was measured using a plate reader

at wavelengths of 570 and 600 nm, taking into account the
background absorbance of both the medium and reagent only
(negative control). Cellular metabolic activity was correlated
TM
with the percentage reduction of AlamarBlue reagent by the
cells, and calculated according to the manufacturers protocol (www.biosource.com). All experiments were conducted in
triplicate.
Overall Phagocytosis Assay
Murine peritoneal macrophages J774 (Lonza, Basel, Switzerland) were seeded in 12-well plates at a concentration of 2
105 cell/mL and were allowed to adhere for 24 h. MPs (Rhodamine labeled) were added to the cells at a concentration of
50 :g/mL (small and big PLGAChi, respectively) and incubated for 24 h at humidified 37 C, 5% CO2 . Moreover, same
amount of cells were seeded on the 25% Pluronic F-127 mixed
with 50 :g/mL of MPs. After 24 h, the culture medium of each
sample in both experiments was collected and the concentration of inflammatory cytokines IL-6 and TNF-" produced by
J774 cells was quantified by ELISA assay (R&D Systems). Cells
were then scraped from the wells, washed three times with cold
phosphate-buffered saline (PBS) to remove unattached particles and analyzed using flow cytometry. SEM was used to visualize cellular interaction with MPs. After allowing 24 h of cellular attachment to glass coverslips at 37 C, cells were fixed with
2.5% glutaraldehyde (SigmaAldrich), washed with increasing
concentrations of ethanol (up to 100%), vacuum dried, and
coated with palladium prior to SEM visualization (Hummer
6.2 Sputtering System; Anatech Ltd., Union City, California).
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RESEARCH ARTICLE Pharmaceutics, Drug Delivery and Pharmaceutical Technology

The MPs were also examined by SEM under a voltage of 5 KV.

After staining cells using a fluorescent WGA and DAPI (Abcam, Cambdridge, UK), cell morphology was evaluated by optical and confocal microscopy. Projection and imaging processing were performed by confocal software (NIS-Element Nikon,
Nikon Instrument Inc., Melville, NY).

v6.02 for Windows. A value of p < 0.05 was considered statistically significant: **p < 0.05; ***p < 0.01.

Flow Cytometry

Poly(lactic-co-glycolic acid)Chi-blended MPs (PLGAChi MPs)

were synthesized as reported in Material and Methods section
and then filtered in order to obtain an enriched population with
size lower than 6 :m (Small PLGAChi) and size bigger of
10 :m (big PLGAChi). The size distribution is reported in
the Supplementary Information (Fig. S1). The presence of a
Chi coating on the MPs surface has been evaluated by SEM
imaging, Z-potential analysis, and FT-IR spectroscopy. Upon
SEM analysis, PLGA particles showed a very smooth surface
while surface topography changes in the PLGAChi were evident (Figs. 1a1d); however, the MPs size distribution was not
affected by the extra Chi layer (Fig. S1). Spectroscopic analysis
of the PLGAChi by FT-IR/ATR (Fig. 1e) confirmed the presence of Chi moieties (18002400 cm1 ); however, the carbohydrates moieties around 1000 cm1 are hidden by the polymeric
vibrational mode.46 Finally, the increase in zeta potential from
40.8 mV (PLGA particles) to +19.5 mV (PLGAChi) with the
introduction of Chi strongly suggests that the polycationic Chi
was adsorbed to the particle surface (Fig. 1e).

The percentage of macrophages that phagocytosed big and

small PLGAChi MPs in the presence or absence of pluronic
F-127 gel was quantified using a BD LSR FortessaTM cell analyzer (Beckton Dickinson, East Rutheford, NJ). To discriminate between big PLGA-MP and cells (macrophages), the
cells were labeled with FITC-conjugated F4/80 (Biolegend, San
Diego, CA) and the percentage of FITC- and PE-positive cells
was evaluated for each sample. A minimum of 10,000 cells
were acquired for each sample and analyzed. Off-line analysis of the files was performed using FCS Express software v.4
(http://www.denovosoftware, Los Angeles, CA).
In Vitro Release of BP
Microspheres of PLGA-BP-Chi (5 mg) and PLGABPChiBP
(5 mg), respectively, were dispersed in PBS (1.5 mL) pH 7, or
in sodium acetate buffer (pH 6.4) at 37 C. At predetermined
time intervals (2461224 h, then daily for 7 days total),
the suspension was centrifuged (4500 rpm; 5 min), the supernatant was collected, and replaced with fresh PBS (1.5 mL).
The amount of BP released at each interval was determined
by analysis of the supernatant using high-performance liquid
chromatography (see Supplementary Informations for details).
Statistical Analysis
All experiments were carried out in triplicates, independently.
The data obtained were expressed in terms of mean standard
deviation values. Wherever appropriate, the data were also
subjected to one-way ANOVA with software GraphPad Prism

RESULTS
Synthesis and Characterization of PLGAChi

Cytotoxicity
Bone-marrow-derived mesenchymal stems cells at step 3 were
incubated with small and big PLGAChi for 1, 3, 5, and 7 days.
At the end of each time point (1, 3, 5, and 7 days), AlamarBlue test demonstrated no difference in mBM-MSC viability
(p > 0.05) from untreated control cells (Fig. 2a). At the end
of 7 days in culture, the cells reached confluency and halted
their growth. The lack of overall MP cytotoxicity on mBM-MSC
populations was complimented by a conspicuous absence of effect on individual MSC cellular morphology by SEM analysis
(Fig. 2b).

Figure 1. Characterization of PLGAChi MPs. SEM imaging of PLGA (a and b) and PLGAChi (c and d). FT-IR spectra of PLGAChi
in comparison with PLGA and bare Chi (e). Z-potential of PLGAChi particles, PLGA, and Chi (f). All the tree characterization techniques
confirmed the presence of Chi on the surface of the MPs.
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DOI 10.1002/jps.24190

RESEARCH ARTICLE Pharmaceutics, Drug Delivery and Pharmaceutical Technology

3727

Figure 2. Cell viability and proliferation. (a) AlamarblueR metabolic assay was used to investigate the differences in metabolic activity of
mBM-MSC grown in the presence of 50 :g/mL of PLGAChi in comparison with control over 7 days of culture (n = 3). Simultaneously, (b) optical
microscope and SEM images of cell culture at different magnification were performed to evaluate cell morphology (IIV). These results show
that the presence of MPs in culture neither affected the growth curve, nor the morphology of the mBM-MSC in comparison with the control
(*p < 0.05).

Figure 3. (a) Flow cytometric analysis shows the uptake of PLGAChi by J774. (a) Cell untreated (I) and cell following the treatment with
(II) small PLGAChi gel, (III) small PLGAChi, (IV) big PLGAChi gel, and (V) big PLGAChi 30 (n = 3). The cells have been labeled by F4/80
(green) and the MPs have been labeled by Rhodamine B (red). The flow analysis shows a decrease of double positive cells that confirm the hiding
effect of Pluronic F-127 gel. (b) SEM images of PLGAChi uptake by J774. Small PLGAChi are completely internalized after 24 h (I) rather
than the big PLGAChi (II).

Macrophage Uptake
After we incubated J774 cells with small and big PLGA
Chi particles loaded with Rhodamine B, the uptake of MPs
by macrophages was measured by flow cytometric analysis
(Fig. 3a). J774 macrophages treated with Rhodamine B-loaded
DOI 10.1002/jps.24190

PLGAChi MPs showed increased granularity over incubation time, strictly dependent on MP size. Twice as many
cells internalized small PLGAChi (12.49%) as compared with
big PLGAChi (6.49%). SEM evaluation confirmed these findings, with small PLGAChi nearly completely internalized by
J774, whereas big PLGAChi appeared to be too large to be
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RESEARCH ARTICLE Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Figure 4. Confocal microscopy images taken at 60x. J774 cells cultured for 24 h were stained for WGA (green), Rodhamine B-labeled PLGAChi
(red) and nuclei (blue). (a) shows small PLGAChi MPs internalized by J774. (b) and (c) are the lateral and three-dimensional projection of the
same picture to confirm the internalization of the MPs, respectively. (df) are the same projection for big PLGAChi.

internalized (Fig. 3b, I and II). Additionally, inclusion of the

MPs inside pluronic F-127 gel significantly diminished uptake
by the macrophages even further. The rate of phagocytosis of
the small PLGAChi/gel was 1.60% and 1.29% for big PLGA
Chi/gel. Figure 4 shows representative confocal images of the
macrophages and MPs after 24 h of incubation. These images
illustrate that small PLGAChi MPs (Figs. 4a4c) are consistently internalized by macrophages; the lateral projection and
three-dimensional reconstruction demonstrate the effectiveness of the encapsulation. Although some cases of opsonization
of Big-PLGAChi (Figs. 4a4c) were witnessed on microscopy,
these were much more sporadic. These results confirm previously reported studies claiming a size-dependent effect of MPs
on macrophage internalization30,47 and the ability of pluronic
F-127 to further reduce phagocytosis.
Inflammatory Reaction
To understand whether our systems maintained the antiinflammatory features of Chi, we evaluated the concentration
of inflammatory cytokines (IL-6 and TNF-") produced by J774
macrophages after exposure to the various MP treatments. Few
prior studies have utilized Chi-coated polymeric MPs and, to
our knowledge, this is the first time that this kind of formulation has been applied for analgesic release. PLGAChi induced
10 times less IL-6 as compared with the uncoated PLGA MPs
(Fig. 5a). The levels of secreted TNF-" in response to big PLGA
Chi displayed a similar trend to IL-6 (Fig. 5b). Moreover, this
effect was even more pronounced when the entire construct is
packaged inside Pluronic F127 gel (Fig. 5). These data clearly
confirm a diminished inflammatory response when MPs are
Taraballi et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:37243732, 2014

coated by Chi and/or included inside another polymer (Pluronic

F127).
Controlled Release
The release rate of BP from our MPs is significantly affected
by differences in local pH as shown in Figure 6a, where the
percentage of drug released into PBS (pH 7) or sodium acetate
buffer (pH 6) is plotted as a function of time. The plot shows
at pH 6 a 2x faster release from the MPs in comparison to pH
7. Embedding MPs in pluronic acid protected from this pHdependent effect to a degree, causing a fourfold slower release
(Fig. 6b). Finally, we compared release from the particles with
BP loaded in the outer Chi layer versus the inner PLGA layer.
The particles with BP loaded in the outer layer displayed a
typical burst release releasing the 100% of the payload after
4 h.

DISCUSSION
Although the presence of at least mild-to-moderate postoperative pain is almost universally expected, improvements in
managing acute pain would significantly improve patient care
and satisfaction, diminish the prevalence of chronic pain syndromes, and ease the overwhelming economic burden that currently plagues society from acute/chronic pain.48 BP is a widely
used amide anesthetic capable of providing local analgesia for
approximately 8 h, but hyperalgesia sets in thereafter once
the drugs effect wanes, thus limiting early functional recovery
and typically necessitating narcotic supplementation for full
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3729

Figure 5. Citokines released by J774 macrophages exposed for 24 h to 0.4 mg/mL of MPs. Inflammatory citokines have been quantified by
ELISA assays for big and small PLGAChi particles outside or inside pluronic gel: IL-6 (a) and TNF-" (b). (n = 6). (**p = 0.05; ***p = 0.01).

analgesia. Enhancing the local delivery of anesthetic would

theoretically limit opioid narcotic need and restore patient
function faster. For this reason, BP has previously been formulated in PLGA MPs to extend its release up to 96 h.49,50
Although such experience with synthetic polymers is extensive
and encouraging, more recent work has shifted toward natural polymers such as alginate and Chi. These alternatives
offer low cost and high biocompatibility with the encapsulation of a wide range of drugs, along with minimal use of organic solvents. Furthermore, bioadhesion, stability, safety, and
existing approval for human use by the FDA are additional
advantages. Chi specifically demonstrates an attractive profile
regarding biodegradability/compatibility, toxicity, hemostasis,
and current use in existing pharmaceuticals.51 To our knowledge, BP-loaded PLGA/Chi MPs have never been prepared and
presented in scientific literature. The purpose of the present
study was to explore polymeric carriers for prolonged local BP
delivery as a potential new analgesic moiety. Encapsulation of
an analgesic inside a carrier greatly prolongs its duration of
action and reduces its systemic toxicity.18 To be successful, the
carrier design must possess: (1) low system toxicity, (2) a tolerable local tissue reaction, and (3) an adequate sustained release
of its payload.
This study proves our ability to reliably synthesize PLGA
ChiBP MPs by the double emulsion method, incorporate a
Chi polymer layer onto the PLGA particle surface, and successfully load and release a local anesthetic from the system (up to
130 mg/mL of MPs/gel). We are able to further control MP
synthesis by size, separating them into small PLGAChi (<6
:m) and big PLGAChi (>10 :m), thus simplifying subsequent
study of their cellular effect to find the ideal size for future in
vivo application. Pertaining to system toxicity, MPs showed no
cytotoxicity or effect on phenotype switching as compared with
controls when applied to mBM-MSC in vitro up to 7 days. Inflammation and foreign body responses are generally viewed as
expected components of the tissue or cellular host responses to
injury during wound healing. Many current medical treatments
DOI 10.1002/jps.24190

employ the implantation of a synthetic substance or device,

which can amplify this natural response. Our in vitro studies using J774 macrophages confirm the absence of immunogenicity of our delivery platform. MPs bigger than 10 :m and
encapsulated in thermoresponsive Pluronic gel are effectively
hidden from macrophage uptake and downregulate the secretion of proinflammatory cytokines (TNF-" and IL-6). The need
for confirmatory in vivo studies is apparent, but this represents
proof-of-concept that PLGAChi Pluronic gel delivery systems
could be an efficient platform to release anesthetic/analgesic
such as BP without causing local tissue side effects.
Currently available clinical formulations of extended-release
BP employ a simple liposomal design that provides analgesia
for roughly 72 h after administration.17,19 A primary aim of this
study was to see whether we could improve upon this using
our advanced delivery system. To that end, we demonstrated
controlled BP release from the platform for up to 7 days. The
versatility of this platform composed of three different layers
PLGA, Chi, and Pluronic gelafforded us the control of BP
release in three distinct temporal peaks corresponding to the
different stages of postoperative pain. The burst release after
5 h from the Chi layer could theoretically cover the immediate
postoperative period after surgery or during a pain crisis. The
release from free particles could provide analgesia up to 4 days,
and finally the embedded MPs in pluronic gel showed a release
kinetic that could last weeks, seeing as though the BP release
was only the 20% of the total loaded drug after 7 days and arrive to 90% after 14 days (Fig. S3). This release profile presents
many advantages that could have great impact if successfully
translated into human clinical care. BP has a long history of
perioperative clinical use, and any extension of its tissue effect
implies potential benefits for ambulatory and inpatient surgical procedures as well as chronic pain management.52 For example, patients commonly cite pain as the primary reason for
presenting to the hospital for readmission, and pain-related impairment is reported to limit ones ability to perform activities
of daily living (e.g., dressing or washing self) for up to 7 days
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Figure 6. Seven-day release kinetics of BP from PLGAChi MPs. (a) BP released at different pH. (b) BP released from PLGAChi MPs
embedded in Pluronic gel. (c) BP released from different layers: outer Chi layer and internal PLGA layer.

postoperatively after uncomplicated ambulatory surgery, making a 7-day BP release particularly promising.53,54 Lastly, it is
known that pH variances exist in normal and inflamed tissue;
and differences in environmental pH alter the duration of local
anesthetic effect as well as degradation rates of polymers.5557
We illustrated that, although a lower pH predictably quickened
drug release from PLGAChi, our delivery systems pluronic gel
carrier improves release consistency via a pH-protective effect.
This feature expands the potential for applying our BP delivery
system brazenly in both normal and inflamed tissues, such as
in wound after surgery.

tion not only seems to be safe and biocompatible, but can be

engineered to be smart enough to avoid speedy immunologic
degradation. Although well-controlled in vivo animal studies
and eventual human trial data are needed to fully substantiate this formulations efficacy, it presents great promise for
improving our ability to provide regional anesthesia while reducing the side effects that come with prolonged opioid narcotic
treatment. This delivery platform is a proof-of-concept for the
new era of engineered pain management therapies.

ACKNOWLEDGMENTS
CONCLUSIONS
These results demonstrate for the first time that it is possible to
encapsulate BP in a gel-based PLGAChi-MP to achieve several
days of prolonged local drug release, and that such a formulaTaraballi et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:37243732, 2014

This work was supported by the Cullen Trust for Health Care
Foundation (project ID: 18130014) and the Brown Foundation
(project ID: 18130011) to Dr. Tasciotti. The work was supported
by Italian Ministry of Health (GR-2010-2318370) and Dr. Corradetti by Fondazione Marche/ISSNAF Post-Doc Fellowship.
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