ELS EV U R
`
`Journal of Controlled Release 712001 5369
`
`journal of
`controlled
`release
`
`wwwelseviercomlocatejconrel
`
`Effects of emulsifiers on the controlled release of paclitaxel
`Taxo1° from nanospheres of biodegradable polymers
`Sishen Feng Guofeng Huang
`and Engineering IMRE 3 Research Link 117602 Sngapore
`of Chemical and Environmental Engineering National University of Sngapore 0 Kent Ridge Crescent
`119260 Sngapore
`
`al nstitute of Materials Research
`
`bDepartment
`
`b
`
`Received
`
`22 September 2000 accepted 24 November 2000
`
`Abstract
`
`Paclitaxel Taxol® is an antineoplastic
`for various cancers especially ovarian and breast cancer Due to its
`drug effective
`such as Cremophor EL has to be used in its clinical administration which causes
`high hydrophobicity however an adjuvant
`the effects of
`serious side effects Nanospheres of biodegradable polymers could be an ideal solution This study investigates
`properties and release kinetics of paclitaxel
`loaded nanospheres
`various emulsifiers on the physicalchemical
`by
`is shown that phospholipids could be a novel
`type of emulsifiers The
`the solvent extractionevaporation
`technique It
`nanospheres manufactured with various emulsifiers were characterized
`their size and size
`by laser
`for
`distribution scanning electron microscopy SEM and atomic force microscopy AFM for their surface morphology
`spectroscopy XPS for their surface
`for their surface charge and most importantly Xray photoelectron
`potential analyser
`chemistry The encapsulation efficiency and in vitro release profile were measured by high performance
`liquid chromatog
`raphy HPLC It
`is found that dipalmitoylphosphatidylcholine DPPC can provide more complete coating on the surface
`of the products which thus results in a higher emulsifying efficiency compared with polyvinyl alcohol PVA Our result
`the chain length and unsaturation
`of the lipids have a significant
`on the emulsifying efficiency
`© 2001 Published by Elsevier Science
`Phospholipids with short and saturated chains have excellent emulsifying effects
`B
`
`light scattering
`
`fabricated
`
`zeta
`
`shows that
`
`influence
`
`Keywords Lipid chain length Lipid chain unsaturation Natural emulsifiers Paclitaxel Single emulsion
`
`1 Introduction
`
`Paclitaxel Taxol® is a diterpenoid extracted from
`the bark of a rare slowly growing Pacific yew or
`Western yew tree Taxus brevifolia Its anti tumor
`in the US National
`activity was detected in 1967
`
`Conesponding
`1936
`
`author Tel +658743835 fax +65779
`
`Email address chefssnusedusg S s Feng
`
`Institute NCI screening of cytotoxic agents
`Cancer
`from natural products and was then found later to be
`drug of special
`a novel promising antineoplastic
`for breast and ovarian cancers pl Its thera
`effect
`peutic efficacy also includes head and neck
`cancer
`small
`colon
`lung cancer
`cell
`cancer multiple
`myeloma melanoma and Kaposis sarcoma
`It was
`approved by US Food and Drug Administration
`FDA for ovarian
`for advanced
`in 1992
`cancer
`in 1994 and for early stage breast
`breast cancer
`
`0168365901$
`see front matter
`PIT S0168365900003643
`
`© 2001 Published by Elsevier Science BY
`
`Abraxis EX2043
`Actavis LLC v Abraxis Bioscience LLC
`1PR201701101 1PR201701103 1PR201701104
`
`

`

`54
`
`Ss Feng G Huang
`
`Journal of Controlled Relea 71 2001 5369
`
`cancer
`
`in October
`
`1999 The action mechanism of
`paclitaxel has been intensively investigated
`
`and the
`
`dissolve
`
`emulsions 1516 and cyclodextrin complexes 17
`Among them some dosage forms can
`shown
`sufficient quantities of paclitaxel and have
`improved anti tumor effects in animal models How
`ever problems such as the in vivo instability of
`liposomes and dose limiting toxicity
`due
`to the
`have also been noticed 1517
`vehicles
`The use of biodegradable polymeric micro nanos
`pheres for controlled delivery of anticancer
`agents
`has advantages in enhancing therapeutic efficacy and
`side effects Certain satisfactory
`have been obtained 1819
`results in clinical
`trials
`has been reported that nanospheres
`In addition it
`show significant advantages over microspheres
`enable
`intravenous
`221 Nanospheres
`injection as
`well as intramuscular and subcutaneous administra
`
`reducing
`
`systemic
`
`reactions
`tion by minimizing possible irritant
`Our group has investigated the feasibility to apply
`polymers as an alter
`nanospheres of biodegradable
`The
`native
`administration system for paclitaxel
`biodegradable polymers used in this research include
`acid PLGA lacticgly
`polyDLlacticcoglycolic
`colic ratio 5050 or 7525 and polyDLlactic acid
`PLA all FDA approved
`The modified solvent
`extractionevaporation technique or the single emul
`sion technique was applied in this study to prepare
`under
`various
`fabrication conditions
`
`nanospheres
`The first
`is to formulate an
`step of this technique
`emulsion The oil phase formed by dissolving the
`drug and the polymer
`in an organic
`solvent
`is
`dispersed in the aqueous phase This is followed by
`of
`solvent Since the oil
`the organic
`evaporation
`drops in the emulsion are easy to aggregate in the
`aqueous phase the emulsifier which is supposed to
`plays an
`phase
`of oil
`aggregation
`the
`
`prevent
`important role in the process
`a wide variety of applications
`Emulsifiers have
`For example in foods they promote the suspension
`of one liquid in another as in the mixture of oil and
`water in margarine shortening ice cream and salad
`dressing Emulsifiers are also used in the preparation
`of cosmetics lotions and certain pharmaceuticals
`where they serve much the same purpose as in foods
`ie they prevent separation of ingredients and extend
`storage life Macromolecular emulsifiers which in
`clude gelatin 23 and PVA polyvinyl alcohol pi
`are widely used in the fabrication of polymeric
`
`micro nanospheres due to their high viscosity in the
`
`leading to
`
`results have suggested that paclitaxel acts to inhibit
`mitosis in tumour cells by binding to microtubules
`Microtubules are made up of tubulins and involved
`in various cellular functions such as cell movement
`nutrition ingestion cell shape control sensory trans
`duction and spindle formation during cell division
`Paclitaxel aids polymerization of tubulin dimers to
`form microtubules and thus stabilizes
`the micro
`tubules The microtubules formed due to paclitaxel
`action are stable and thus dysfunctional
`cell death 241
`There are two limitations for clinical application
`of paclitaxel One is its availability Although ex
`has
`increased
`traction of paclitaxel
`in large scale
`yields to 004 four trees have
`to be sacrificed to
`produce 2 g of the drug for the chemotherapy of one
`patient This is not affordable from an environmental
`point of view Although having been achieved
`full
`synthesis is not practical as more than 200 steps are
`needed and the price is thus too high An appropriate
`solution seems to be a semisynthesis which extracts
`paclitaxel from needles and twigs of more abundant
`English yew trees or Chinese red bean yew trees
`Another limitation is its difficulty in clinical adminis
`in
`tration Paclitaxel
`is highly hydrophobic water
`05 mg1 An ad
`soluble with water solubility
`of Cremophor EL polyox
`consisting
`juvant
`yethylated castor oil and dehydrated alcohol has to
`be used in current clinical administration of paclitax
`has been
`el which causes
`serious
`side effects It
`believed that side effects caused by Cremophor EL
`include hypersensitivity reactions nephrotoxicity and
`It was also reported that Cremophor
`neurotoxicity
`EL has influence on the functions of endothelial
`and
`vascular muscle and causes vasodilation laboured
`breathing lethargy and hypotension In application
`leaching of diethylhexylphthalate DEHP
`from plasticized polyvinyl chloride PVC containers
`infusion line 510
`and intravenous
`Due to the limitation of paclitaxel
`administration its dosage and infusion period have
`thus been restricted Best clinical effects have not
`been achieved yet Alternative dosage forms are thus
`necessary to overcome the problem caused by Cre
`mophor EL Such alternative
`dosages may include
`liposomes 1113 mixed micelles 14 parenteral
`
`it causes
`
`in its clinical
`
`

`

`Ss Feng G Huang
`
`Journal of Controlled Relea 71 2001 5369
`
`55
`
`around the
`
`formation of the micro nanospheres It
`
`is found that
`
`aqueous solution and strong adsorption
`emulsion drops However
`these macromolecules
`not easily washed out of the micro nanospheres 25
`As a result
`they may cause troubles in purification
`of products and thus influence the properties of the
`formed micro nanospheres Among various natural
`emulsifiers phospholipids have been widely used as
`wetting and emulsifying agents and for other pur
`The products
`in which phos
`poses in industry
`pholipids are used as emulsifiers include
`animal
`and mixes chocolate
`feeds baking
`products
`metics and soaps
`dyes insecticides paints
`plastics However
`their application as emulsifiers in
`the solvent extractionevaporation technique to fabri
`
`are
`
`cos
`and
`
`suggested
`
`the
`
`It
`
`is
`
`to
`
`cate polymeric micronanospheres as a drag delivery
`system has rarely been reported Only a few publi
`cations
`the
`of 12dipal
`use
`that
`mitoylphosphatidylcholine DPPC as an additive
`may be able to improve the performance of
`in blood flow 26
`produced PLGA microspheres
`the pulmonary absorption of peptides and
`enhance
`27 and reduce phagocytic
`the
`uptake of
`proteins
`microspheres 28 All
`the po
`these facts hint at
`tential application of phospholipids as natural emul
`sifiers in the double or single emulsion technique to
`fabricate polymeric micro nanospheres
`therefore
`the emphasis of
`this paper
`this new type of emul
`recognize and characterize
`sifier We shall prove that compared with the tradi
`emulsifiers such as PVA phos
`chemical
`in emulsion stabili
`pholipids are of higher efficiency
`in polymeric micronanos
`zation and can result
`of
`better
`physicalchemical
`
`tional
`
`pheres
`
`properties
`
`Characterization
`
`of
`
`paclitaxelloaded
`
`polymeric
`
`compared with traditional chemical emulsifiers such
`as polyvinyl alcohol PVA phospholipids can pro
`vide a more complete coating on the surface of the
`products which thus results in a higher emulsifying
`efficiency Phospholipids of various chain length and
`chain
`unsaturation
`such
`as 12didecanoylcphos
`
`phatidylcholine
`
`phatidylcholine
`
`12dipalmitoylphos
`12 distearoylpho s
`12dioleoylphos
`for their
`
`DDPC
`DPPC
`phatidylcholine DSPC and
`phatidylcholine DOPC are
`investigated
`emulsifying effects in the single emulsion process in
`The
`loaded
`fabrication of paclitaxel
`nanospheres
`result shows that
`the length and unsaturation of the
`on the emul
`influence
`lipid chains have significant
`and
`sifying efficiency Phospholipids with short
`saturated chains such as DDPC can have excellent
`emulsifying effects
`
`2 Materials and methods
`
`21 Materials
`
`Paclitaxel Taxol® is the product of Dabur India
`India or Hande Biotechnology China Polymers
`acid PLGA
`such
`as polyDLlacticcoglycolic
`ratio of 7525 and 5050 MW=
`with LG molar
`acid PLA
`90000120000
`and polylactic
`such as polyvinyl alcohol PVA MW=
`chemicals
`3000070000 and methylene chloride DCM and
`
`phatidylcholine
`
`phatidylcholine
`
`various
`
`lipids
`
`such
`
`as
`
`12didecanoylcphos
`
`DDPC
`12dipalmitoylphos
`DPPC
`12 distearoylpho s
`phatidylcholine DSPC and
`12dioleoylphos
`phatidylcholine DOPC were all purchased from
`Sigma St Louis MO
`
`22 Methods
`
`221 Preparation of polymeric nanospheres
`Paclitaxel and 200 mg of PLGA were dissolved in
`chloride DCM The formed
`16 ml of methylene
`solution was
`added to
`250 ml of
`subsequently
`distilled water The resulting emulsion was sonicated
`with an energy output of 50 W in a pulse mode for
`90 s The oil inwater emulsion was then stirred
`room temperature with the magnetic
`
`overnight at
`
`emulsifiers is
`nanospheres fabricated with various
`conducted by laser light scattering for their size and
`electron
`
`size distribution
`
`scanning
`
`microscopy
`
`SEM and atomic force microscopy AFM for their
`surface morphology and Xray photoelectron
`spec
`troscopy XPS for the chemical
`structure of
`surface The drug encapsulation efficiency and the in
`are measured by high per
`formance liquid chromatography HPLC Except for
`recognition of phospholipids as effective emulsifiers
`
`vitro release kinetics
`
`their
`
`another feature of this paper is its use of atomic force
`microscopy AFM and Xray photoelectron
`spec
`troscopy XPS to characterize
`the prepared nanos
`pheres and to manifest the emulsifiers role in the
`
`

`

`56
`
`Ss Feng G Huang
`
`Journal of Controlled Relea 71 2001 5369
`
`stirrer
`
`chloride The pro
`to evaporate methylene
`duced nanospheres were collected by centrifugation
`12000 rpm 30 mm and washed with de ionized
`times to remove excessive emulsifiers
`four
`water
`The product was freezedried to get
`
`fine powders
`
`were
`
`222 Particle characterization
`Particle size and size distribution were measured
`light scattering with particle size analyzer
`by laser
`Instruments Huntsville NY
`90 Plus Brookhaven
`USA For the measurement 2 mg nanospheres
`in de ionized water which was
`dispersed
`filtered before use
`Dried nanospheres were coated with gold for 3
`electron microscopy SEM
`min before
`scanning
`Hitachi S4100 Japan was applied The accelerating
`voltage ranged from 5 to 15 kV during scanning
`The nanospheres were fixed on double sided sticky
`force microscopy AFM was
`atomic
`tape before
`Thereafter AFM images were obtained
`conducted
`Instrument Santa Bar
`by Nanoscope
`IIIa Digital
`bara CA USA in tapping mode The cantilever
`oscillated at its proper frequency 300 KHz and the
`driven amplitude was 130 mV
`The zeta potential of the nanospheres was mea
`analyzer Zeta Plus
`zeta potential
`sured by a
`Instruments Huntsville NY USA
`Brookhaven
`First 3 mg nanospheres were dispersed in 10 ml
`buffer solution with different pH value which was
`followed by sonication for 3 min The zeta potential
`of products was measured with palladium electrodes
`and the mean of five readings was taken
`spectroscopy XPS Axis
`For Xray photoelectron
`His a product
`from Kratos Analytical with mag
`nesium as the node was used For all samples the
`survey spectrum recorded covered a binding energy
`range from 0 to 1200 eV using a pass energy of 80
`eV Curve fitting was performed using the software
`supplied by the manufacturer
`
`223 In vitro release of paclitaxel
`Typically 10 mg of nanospheres were dispersed in
`phosphate buffered saline PBS the pH of which
`was maintained at 74 The buffer solution was kept
`in an orbital shaker
`that was vibrated
`at a constant
`rpm and the temperature was
`rate of 166
`kept
`constant at 372°C At given time intervals three
`
`tubes of each
`
`formula of nanospheres were with
`at 12000 rpm for 5 min The
`drawn and centrifuged
`and resus
`nanospheres were
`taken
`precipitated
`pended in 10 ml of fresh release medium and placed
`back in the shaker while the supernatant solution
`was kept
`for high performance
`liquid chromatog
`raphy HPLC analysis In order to examine the
`in the supernatant
`solution the
`paclitaxel content
`following procedure was adopted Paclitaxel
`in the
`release medium firstly extracted with 1 ml of DCM
`in 1 ml of mobile phase acetoni
`was reconstituted
`trilewater 11 DCM was then evaporated under a
`stream of nitrogen For HPLC analysis the C18
`column was used and the mobile phase was delivered
`1 mlmin A total of 100 µ1 of sample
`at a rate of
`was injected with an auto sampler and the column
`effluent was detected at 227 nm with an ultra violet
`UV detector
`
`efficiency
`
`224 Encapsulation
`First 3 mg of paclitaxelloaded
`nanospheres
`were dissolved in 1 ml DCM and 9 ml of mobile
`phase subsequently were added to the solution A
`nitrogen stream was introduced to evaporate DCM at
`room temperature until a clear solution was obtained
`The resulting solution was analyzed by HPLC in the
`conditions mentioned
`above
`
`the
`
`225 Extraction factor and recovery efficiency
`measurement
`Due to inefficient extraction the extraction factor
`had to be analyzed
`for calibration The procedure
`was as follows 29 Paclitaxel of known concen
`tration in aqueous solutions varying from 005 to 05
`mg1 was extracted
`and its
`was
`concentration
`investigated by HPLC The results showed that
`solution contained 16 of
`extracted
`the original
`amount of
`the paclitaxel Therefore the in vitro
`release result determined by this method should be
`corrected by 16 due to inefficient extraction
`in DCM
`Similarly a known amount of paclitaxel
`was subjected to the procedure mentioned
`above
`The result showed that 78 of paclitaxel
`remained
`after these procedures Consequently 78 was used
`obtained from
`
`to correct
`
`the encapsulation efficiency
`the method mentioned above
`
`

`

`Ss Feng G Huang
`
`Journal of Controlled Relea 71 2001 5369
`
`57
`
`a
`
`0
`
`CH3
`
`II
`
`CH2C0
`
`t
`
`bC
`
`H2CHt CH2CH1 00n
`
`OH
`
`0=C CH3
`
`CH 214 CH 3
`
`II
`
`C 0
`H2C C
`1 0
`HC C 0 CH 214 CH 3
`0
`HH2C 0 P 0 CC N+CH33
`
`H 2 H 2
`
`II
`
`0
`
`d
`
`9
`
`0
`
`HC
`CH
`
`H H C
`0
`
`3
`
`OH
`
`Fig 1 Chemical structures of a PLGA b PVA c DPPC and
`d paclitaxel
`
`1
`
`represents the carbon in CC or CH Peak 2 is
`to hydroxyl COH
`
`generated by the carbon next
`Peak 3 is contributed by the carbon of ester Peak 4
`to carbon
`Since the
`in carboxylate
`contribution of three carbons peaks 1 3 and 4 is
`close in the molecular chain of PLGA the per
`of
`these three carbons
`should vary little
`centages
`from one to another This expectation agrees with the
`quantification report summarized in Table 1 How
`ever this is not the case for pure PVA because of the
`different chemical structures Compared with PLGA
`carbon of ester peak 3 present
`there was no
`PVA instead peak
`suggests the existence of
`
`corresponds
`
`2
`
`in
`
`3 Results and discussion
`
`First we outline the effects of the various emul
`sifiers used in the single emulsion process on the
`physicalchemical properties of the produced nanos
`The influence of
`the traditional synthetic
`pheres
`emulsifiers such as PVA and the natural emulsifiers
`such as DPPC is compared This will
`then be
`the effects of
`followed by a close examination of
`structure of the phospholipid used on the
`chemical
`The em
`of
`the prepared
`properties
`nanospheres
`phasis in the present study will be on the effects of
`and unsaturation
`chain
`of
`the
`the alkyl
`length
`phospholipids We want
`to determine the best of the
`lipids which have
`the best emulsifying
`various
`effects and thus result in polymeric nanospheres of
`the best physical and chemical properties and release
`profile for clinical administration of paclitaxel The
`can also be applied
`to the
`technique
`developed
`general micronanosphere system to deliver other
`anticancer drugs such as adriamycin camptothecin
`irinotecan and antimalarial drugs such
`fluorouracil
`and lumifantrine as well
`as halofantrine
`
`31 Effects of various emulsifiers
`
`311 Surface chemistry
`Surface chemistry of the nanospheres prepared by
`technique was
`the solvent
`
`analysed
`
`photoelectron
`
`spectroscopy
`
`extractionevaporation
`by Xray
`XPS From the chemical structures of PLGA PVA
`DPPC and paclitaxel
`illustrated in Fig 1 it
`is
`is the only substance which
`that paclitaxel
`apparent
`contains nitrogen in the nanospheres prepared with
`PVA as emulsifier Therefore nitrogen can be the
`characteristic element of paclitaxel As illustrated in
`Fig 2 the scan of nitrogen failed to detect
`existence of Nis atomic
`is of nitrogen
`core level signal on the exterior This clearly indi
`there was little paclitaxel on the surface
`cated that
`This fact may be attributed to the very low solubility
`in water which makes the drug tend to
`of paclitaxel
`stay inside polymeric nanospheres rather than diffuse
`to the surface
`The XPS Cis atomic orbital
`is of carbon regions
`were then studied The results displayed
`in Fig 3
`in the analysis of the pure PLGA 7525
`show that
`and PVA a total of four peaks were presented Peak
`
`orbital
`
`the
`
`

`

`Ss Feng G Huang
`
`Journal of Controlled Relea 71 2001 5369
`
`58
`
`PLGA rtano0
`
`mulsecr 1114
`
`chemistry analysis of PLGA nanospheres
`Fig 2 Surface
`fabri
`cated with PVA as emulsifier Nis with Xray photoelectron
`spectroscopy
`
`hydroxyl carbon in the molecule In addition the
`quantification report shows that carbon content of
`carboxylate was very low implying a very low
`degree of esterification of polyvinyl alcohol
`Compared with pure PLGA and PVA paclitaxel
`loaded PLGA nanospheres fabricated with PVA as
`emulsifier demonstrated the existence of all
`the four
`to 4 Fig 4 Furthermore the
`carbons from peak 1
`atomic composition of these carbons lies between the
`for pure PLGA and PVA which
`values determined
`strongly suggested the co existence of both PLGA
`and PVA on the surface
`The discussion
`so far has concentrated
`
`on the
`surface property of nanospheres manufactured with
`PVA as emulsifier To conclude
`the points discussed
`that both PLGA and PVA ex
`above
`demonstrated
`isted on the exterior of the nanospheres prepared
`with PVA as emulsifier
`In order to compare the
`effect of emulsifiers on the surface property the P2p
`atomic orbital 2p of phosphorous and Cis atomic
`is of carbon regions of the surface of the
`nanospheres prepared with DPPC as emulsifier were
`characterized by XPS as well Fig 5 First of all
`the scan of phosphorous P2p peak shown in Fig
`5a suggests the existence of phosphorus
`on the
`of DPPC
`From the chemical
`surface
`is obvious that only DPPC
`demonstrated in Fig 1 it
`phosphate includes phosphorus Therefore phosphor
`element of DPPC in this
`us can be a characteristic
`system As a result
`the existence of P2p peak
`indicated the distribution of DPPC on the surface
`
`orbital
`
`structures
`
`Binting orV
`
`Fig 3 Surface chemistry analysis of Cis with Xray photoelec
`tron spectroscopy a Polylactidecoglycolide PLGA b
`polyvinyl alcohol PVA and c DPPC
`
`of Cis was also performed sub
`Investigation
`to closely examine the surface chemistry
`
`sequently
`
`nanospheres
`of
`In contrast
`to the
`the prepared
`nanospheres prepared with PVA as emulsifier the
`XPS spectra of the nanospheres prepared with DPPC
`
`

`

`Ss Feng G Huang
`
`Journal of Controlled Relea 71 2001 5369
`
`59
`
`C0C=0
`
`3033
`
`2141
`
`386
`
`2175
`
`OC=0
`
`3118
`
`481
`
`345
`
`2010
`
`520
`
`COH
`
`4647
`
`2844
`
`a
`
`PLGA nanefipieleg
`E mkrer NPR
`
`134
`
`132
`
`130
`
`Binding Energv eV
`
`b P
`
`LGA
`pr
`Emuktifter WPC
`
`Table 1
`XPS Cis analysis of PLGA nanospheres
`
`Sample
`
`XPS Cis envelope ratios
`CCCH
`
`PLGA
`PVA
`DPPC
`PLGAPVA nanospheres
`PLGADPPC nanospheres
`
`3839
`
`4872
`
`7514
`
`4761
`
`7305
`
`that
`
`the ratio of carbon
`
`aliphatic
`
`showed a sharply different shape which implied a
`varied surface chemistry caused by emulsifiers Fig
`5b The quantification report Table 1 confirmed
`groups of
`in methylene
`carbon in the DPPC
`chains to carboxylate
`containing nanospheres 730552 was much higher
`the PVA containing
`than
`that of
`nanospheres
`47612010 although
`the
`surface of both the
`nanospheres was dominated by carbon in aliphatic
`carbon chain Besides a comparison
`between
`the
`XPS results of PLGA nanospheres prepared with
`illustrated in Fig 5b and
`DPPC as emulsifier
`those of pure DPPC presented in Fig 3c shows
`they bore a striking resemblance each to other
`also indi
`Furthermore the quantification analysis
`the position and area of peaks of DPPC
`cates
`emulsified nanospheres were very close to those of
`pure DPPC All
`these results
`revealed
`the
`surface of DPPC emulsified nanospheres was domi
`nated by DPPC
`
`that
`
`that
`
`that
`
`chemistry analysis of PLGA nanospheres
`Fig 4 Surface
`fabri
`cated with PVA as emulsifier Cis with Xray photoelectron
`spectroscopy
`
`Fig 5 Surface chemistry analysis PLGA nanospheres
`fabricated
`with DPPC as emulsifier with Xray photoelectron spectroscopy
`a P2p region b Cis region
`
`Binding EntrgyiV
`
`

`

`60
`
`Ss Feng G Huang
`
`Journal of Controlled Relea 71 2001 5369
`
`oil
`
`that
`
`that DPPC is dominant on the surface of
`The fact
`the DPPC emulsified nanospheres
`indicated
`DPPC functioned
`as an emulsifier at
`the interface
`between
`and water phases in the preparation
`accounted
`for the high
`This phenomenon
`process
`encapsulation efficiency of the nanospheres prepared
`with DPPC as emulsifier as will be shown below In
`addition it has been shown that coating of DPPC on
`the nanosphere surface can improve the flow proper
`ty and phagocytal behaviour of the produced nanos
`pheres 2728 which suggests the great advantage
`of DPPC as emulsifier
`in the solvent evaporation
`to prepare micro nanospheres as a drug
`delivery system
`
`technique
`
`312 Surface morphology
`Besides surface chemistry surface morphology is
`another crucial property for the prepared nanos
`loaded PLGA
`pheres The morphology of paclitaxel
`fabricated with PVA and DPPC as
`nanospheres
`emulsifiers respectively was examined by scanning
`electron microscopy SEM and the result is shown
`in Fig 6 from which it
`can be seen
`the
`that
`nanospheres appeared to be round in shape and the
`surface was smooth The size of nanospheres emul
`sified with PVA ranged from 300 nm to 1 µm while
`the most frequent size of nanospheres was 700 nm
`the DPPC emulsified nanospheres dem
`In contrast
`onstrated a relatively smaller size
`the prepared
`Nevertheless
`nanospheres
`are
`for SEM to investigate
`due to its
`small
`closely
`limited magnification Therefore atomic force micro
`scopy AFM was employed in our
`research to
`the detailed morphology of
`the study of
`promote
`nanospheres The advantage of this approach is that
`
`too
`
`it can reveal
`the structure with very high resolution
`as shown in Figs 711
`Fig 7a gives an overview of a typical nanos
`phere with a diameter of 500 nm and its surface
`seemed to be rather smooth However
`this was to
`some extent misleading because a certain degree of
`the superficies was noticeable
`in a
`roughness of
`investigation of its coronal as shown in Fig
`
`7b
`The sectioning
`the nanospheres are
`analyses of
`presented in Figs 9 and 10 while the results that
`emerged from magnification analysis of the nanos
`pheres are given in Fig 11 These results clearly
`
`closer
`
`Fig 6 SEM images of PLGA nanospheres with a PVA and b
`DPPC as emulsifiers
`
`substantiated the existence of complex topography of
`the nanosphere which contained
`some micro caves
`and pores on the surface rather
`than simply smooth
`morphology The nanospheres fabricated with DPPC
`as emulsifier were similar This may be evidence of
`can also be con
`diffusion release mechanism It
`cluded from these images
`fabricated
`with
`
`that
`
`the surface of
`the
`DPPC
`was much
`nanospheres
`smoother
`the nanospheres
`than that of
`with PVA This
`can be explained
`packing of DPPC on the surface
`
`fabricated
`
`by the denser
`
`It
`
`313 Size and encapsulation
`efficiency
`to examine the effect of various
`is important
`emulsifiers on the size size distribution and encapsu
`of the manufactured
`nanospheres
`lation efficiency
`The average size of the nanospheres characterized by
`and their encapsulation ef
`laser
`
`light
`
`scattering
`
`

`

`Ss Feng G Huang
`
`Journal of Controlled Relea 71 2001 5369
`
`61
`
`b
`
`11141
`
`100
`
`300
`
`400
`
`Fig 8 Topography height analysis of nanospheres
`with DPPC as emulsifier by atomic force microscopy
`
`prepared
`
`Such an explanation ag
`the fabrication procedure
`is thus understand
`rees with the results of Gorner It
`
`results
`
`sifier
`
`size distribution
`
`efficiency
`
`able that
`the encapsulation efficiency of nanospheres
`is usually lower than that of microspheres 2332
`showed that compared
`Nevertheless
`our
`with the nanospheres fabricated with PVA as emul
`those prepared with DPPC had similar size and
`but much
`higher encapsulation
`This suggested that DPPC could be a
`than PVA to fabricate
`better emulsifier
`polymeric
`micro nanospheres in the solvent extractionevapo
`ration technique
`a high concentration
`Obviously
`leads to a reduced size of the produced nanospheres
`This can be expected from the function of emulsifier
`as emulsion stabilizer to form nanospheres
`is easy
`insufficient amount of the emul
`sifier would fail
`the nanospheres and
`to stabilize all
`thus some of them would tend to aggregate As a
`result nanospheres with large size would be pro
`In the case of DPPC since the fabricated
`duced
`nanospheres can be completely covered by the DPPC
`even at
`low concentration
`as proved by
`molecules
`the XPS results demonstrated above the surplus of
`DPPC does not help to further reduce the size of
`
`of emulsifier
`
`It
`
`to understand that
`
`LIM
`
`4E61
`
`500
`
`200
`
`300
`
`100
`
`400
`
`Fig 7 Topography height analysis of nanospheres
`with PVA as emulsifier by atomic force microscopy
`
`prepared
`
`ficiency EE measured by HPLC are summarized in
`Table 2 From this table it
`the EE of
`the nanospheres prepared with the solvent extraction
`
`is obvious that
`
`evaporation technique ranged from 20 to 50 which
`is close to that reported by Gorner 30 However all
`these values are lower than those of the microspheres
`prepared by Wang 23 It has been reported that
`the
`EE of the nanospheres increases with the increment
`of their diameter 3031 This can be explained by
`that an increase of the diameter results in a
`the fact
`decrease of the surface area per unit volume which
`reduce the possibility of drug loss
`will consequently
`by diffusion towards the suspending medium during
`
`nanospheres
`The amount of emulsifier plays a fundamental
`in determining not only the size of fabricated nanos
`as well The
`too much
`
`of
`
`this study
`
`indicated
`
`that
`
`the encapsulation
`
`efficiency
`
`role
`
`pheres but
`outcome
`
`

`

`62
`
`S s Fang G Huang
`
`Journal of Controlled Releaw 71 2001 5369
`
`Section
`
`is
`
`050
`
`JIM
`
`100
`
`200
`
`400
`
`Fig 9 Crosssection analysis of nanospheres
`as emulsifier by atomic force microscopy
`
`prepared with PVA
`
`emulsifier both PVA and DPPC would result in a
`reduced encapsulation efficiency of the nanospheres
`This can possibly be explained by the fact
`that some
`drug molecules may bind to the excessive emulsifier
`molecules and thus cause
`the loss of drugs in the
`fabrication process It can thus be concluded that
`the
`presence of an optimal amount of the emulsifier used
`for
`in the fabrication process
`the
`encapsulation
`is justifiable This finding is applicable not
`efficiency
`only to nanospheres but also to microspheits
`Another point to be noted from Table 2 is that
`the
`emulsifier DPPC resulted a much higher emulsifying
`than PVA This implies that much lower
`efficiency
`amount of DPPC 140 of PVA is needed in order
`
`Fig 10 Crosssection analysis of nanospheres
`DPPC as emulsifier by atomic force microscopy
`
`prepared with
`
`to achieve
`nanospheres with similar encapsulation
`efficiency This is quite possible since DPPC has
`tendency to migrate to the surface of nanos
`greater
`has been revealed by our XPS
`pheres This
`analysis of the surface chemistry It
`is the emulsifier
`on the surface of nanospheres that
`functions
`as the
`effective emulsifier to prevent aggregation and stabi
`lize the emulsion
`
`fact
`
`314 In vitro release profile
`The most important effect of emulsifiers on the
`properties of the produced nanospheres is that on the
`release behaviour which will be presented in this
`
`

`

`Ss Feng G Huang
`
`Journal of Controlled Relea 71 2001 5369
`
`63
`
`a
`
`0
`
`02
`
`01 U
`
`00 U
`
`112
`n n00 11
`
`40
`
`020
`
`020
`
`GAD
`
`040
`
`020
`
`010
`
`i1u
`
`020
`
`030
`
`Fig 11 Topography amplitude analysis of polylacticcogly
`colic PLGApaclitaxel
`by atomic
`characterized
`force microscopy a PVA b DPPC as emulsifier
`
`nanospheres
`
`the in vitro release profile of
`section Obviously
`fabricated with DPPC as emulsifier
`nanospheres
`shown in Fig 12 was similar to that prepared with
`PVA that
`is after an initial burst paclitaxel was
`released
`rate In addition
`at a nearly constant
`
`Table 2
`
`efficiency and mean size of PLGA nanospheres
`prepared by different emulsifiers with varied concentration
`Mean size nm
`9735±410
`8010 ±380
`5710±890
`6330±1340
`
`The encapsulation
`
`Emulsifier
`
`PVA 2 wt
`PVA 4 wt
`DPPC 005 wt
`DPPC 01 wt
`EE encapsulation
`
`EEa
`
`402
`
`229
`
`449
`
`340
`
`efficiency
`
`Release profile of PLGA rolcrosphere
`
`Reline titne dityi
`
`Fig 12 The in vitro release profile of PLGApaclitaxel
`pheres with DPPC and PVA as emulsifier
`
`nanos
`
`nanospheres produced with DPPC as emulsifier had a
`relatively lower release rate which meant a longer
`duration of release
`
`is
`
`first
`
`Since the degradation time of polymers is usually
`more than 3 months its effect on the release in the
`2 months
`less significant Therefore the
`dominant
`release mechanism during this period
`might be diffusion alone In addition the AFM
`investigation has shown the existence of micro caves
`and micro pores
`on the surface of nanospheres
`which provided experimental evidence for the diffu
`sion release mechanism In general
`the release by
`diffusion is greatly dependent on the surface mor
`the product As the AFM results have
`phology of
`PVA emulsified nanospheres showed
`demonstrated
`more pores on the surface than DPPC emulsified
`nanospheres Consequently nanospheres fabricated
`with DPPC produced a lower release rate and thus
`longer release period
`
`32 Effect of chain length and chain unsaturation
`of
`
`lipids
`
`The discussion
`above has pointed
`out clearly that phospholipids can act as efficient
`
`demonstrated
`
`emulsifiers in the solvent
`extractionevaporation
`to fabricate micro nanospheres for con
`
`technique
`
`trolled drug delivery purpose It
`that
`is apparent
`structure of the lipids such as head group
`chemical
`type chain length and existence of double bonds in
`
`

`

`64
`
`Ss Feng G Huang
`
`Journal of Controlled Relea 71 2001 5369
`
`role in the
`
`the alkyl chain can play an important
`and the properties
`emulsifying efficiency
`manufactured